Compositions and methods for modified b cells expressing reassigned biological agents

ABSTRACT

Compositions and methods are disclosed herein for producing one or more immunoglobulins in an isolated cytotoxic B lymphocyte cell line. An isolated cell line includes an isolated B lymphocyte cell line capable of expressing at least one exogenously incorporated membrane immunoglobulin capable of binding to a first antigen and at least one endogenous secreted immunoglobulin capable of binding to a second antigen, and further capable of expressing at least one exogenously incorporated recombinant B cell receptor that signals for expression of cytotoxic effector molecules.

If an Application Data Sheet (ADS) has been filed on the filing date ofthis application, it is incorporated by reference herein. Anyapplications claimed on the ADS for priority under 35 U.S.C. §§ 119,120, 121, or 365(c), and any and all parent, grandparent,great-grandparent, etc. applications of such applications, are alsoincorporated by reference, including any priority claims made in thoseapplications and any material incorporated by reference, to the extentsuch subject matter is not inconsistent herewith.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application claims the benefit of the earliest availableeffective filing date(s) from the following listed application(s) (the“Priority Applications”), if any, listed below (e.g., claims earliestavailable priority dates for other than provisional patent applicationsor claims benefits under 35 USC § 119(e) for provisional patentapplications, for any and all parent, grandparent, great-grandparent,etc. applications of the Priority Application(s)).

PRIORITY APPLICATIONS

The present application constitutes a continuation-in-part of U.S.patent application Ser. No. 15/178,715, entitled COMPOSITIONS ANDMETHODS INCLUDING CYTOTOXIC B LYMPHOCYTE CELL LINE EXPRESSING EXOGENOUSMEMBRANE IMMUNOGLOBULIN DIFFERENT FROM SECRETED IMMUNOGLOBULIN, namingRoderick A. Hyde, Wayne R. Kindsvogel, and Gary L. McKnight asinventors, filed 2016 Jun. 10, with attorney docket no.0810-002-002-CIP001, which is currently co-pending or is an applicationof which a currently co-pending application is entitled to the benefitof the filing date, and which is a continuation-in-part of U.S. patentapplication Ser. No. 14/549,685, now U.S. Pat. No. 9,512,213, entitledCOMPOSITIONS AND METHODS INCLUDING RECOMBINANT B LYMPHOCYTE CELL LINEINCLUDING AN EXOGENOUSLY INCORPORATED NUCLEIC ACID EXPRESSING ANEXOGENOUS MEMBRANE IMMUNOGLOBULIN REACTIVE TO A FIRST ANTIGEN ANDINCLUDING AN ENDOGENOUS GENE EXPRESSING AN ENDOGENOUS SECRETEDIMMUNOGLOBULIN REACTIVE TO A SECOND ANTIGEN, naming Roderick A. Hyde andWayne R. Kindsvogel as inventors, filed 21 Nov. 2014 with attorneydocket no. 0810-002-002-000001, and which is a continuation of U.S.patent application Ser. No. 13/374,351, now U.S. Pat. No. 9,175,072,entitled COMPOSITIONS AND METHODS INCLUDING RECOMBINANT B LYMPHOCYTECELL LINE INCLUDING AN EXOGENOUSLY INCORPORATED NUCLEIC ACID EXPRESSINGAN EXOGENOUS MEMBRANE IMMUNOGLOBULIN REACTIVE TO A FIRST ANTIGEN ANDINCLUDING AN ENDOGENOUS GENE EXPRESSING AN ENDOGENOUS SECRETEDIMMUNOGLOBULIN REACTIVE TO A SECOND ANTIGEN, naming Roderick A. Hyde andWayne R. Kindsvogel as inventors, filed 22 Dec. 2011 with attorneydocket no. 0810-002-002-000000.

If the listings of applications provided above are inconsistent with thelistings provided via an ADS, it is the intent of the Applicant to claimpriority to each application that appears in the DomesticBenefit/National Stage Information section of the ADS and to eachapplication that appears in the Priority Applications section of thisapplication.

All subject matter of the Priority Applications and of any and allapplications related to the Priority Applications by priority claims(directly or indirectly), including any priority claims made and subjectmatter incorporated by reference therein as of the filing date of theinstant application, is incorporated herein by reference to the extentsuch subject matter is not inconsistent herewith.

SUMMARY

Compositions and methods are disclosed herein for producing animmunoglobulin in a recombinant B lymphocyte cell line. Compositions andmethods are disclosed herein for treating a disease in a vertebratesubject with an immunotherapeutic product. The immunotherapeutic productcan include the recombinant B lymphocyte cell line that produces one ormore antibodies. The immunotherapeutic product can include therecombinant B lymphocyte cell line that is an exceptional antigenpresenting cell.

Compositions and methods are disclosed for providing a therapeuticallyeffective amount of one or more modified B lymphocytes to a patientsuspected or known to have a disease, disorder, or condition of theimmune system including, but not limited to, infectious disease,auto-immune disease, cancer, neuro-physiological disease or disorders,or other pathological conditions. In an embodiment, a cohort of modifiedB lymphocytes is provided, as described herein. In an embodiment, amonoclonal administration of modified B lymphocytes is provided, asdescribed herein. In an embodiment, a polyclonal administration ofmodified B lymphocytes is provided, as described herein.

Compositions and methods are disclosed herein for producing one or moreimmunoglobulins in an isolated modified B cell that may be part of a Blymphocyte cell line. Compositions and methods are disclosed herein forproducing one or more immunoglobulins in the isolated modified B cell orB lymphocyte cell line that direct cell signaling by membraneimmunoglobulin in the isolated modified B cell or B lymphocyte cellline. Immune cell therapy in a vertebrate subject can includeadministering to the vertebrate subject the isolated modified B cell orB lymphocyte cell line that synthesizes secreted immunoglobulins andmembrane immunoglobulins each having different target antigens. Immunecell therapy in a vertebrate subject can include administering to thevertebrate subject antigen presenting cells comprised of the isolatedmodified B cell or B lymphocyte cell line that direct antigeninternalization and processing to produce exceptional antigen presentingcells. The isolated modified B cell or B lymphocyte cell line canproduce antigen presenting cells that are exceptional or superior atcapturing, internalizing and presenting the antigen recognized by theendogenous or exogenously derived membrane immunoglobulin. Compositionsand methods are disclosed herein for treating a disease in a vertebratesubject with an immunotherapeutic product. The immunotherapeutic productcan include the isolated modified B cell or B lymphocyte cell linehaving an endogenously-derived or exogenously derived membraneimmunoglobulin reactive to a first antigen (e.g., capable of binding toa first antigen) wherein the isolated modified B cell or B lymphocytecell line produces one or more secreted immunoglobulins reactive to asecond antigen or produces a reassigned biological agent. Theimmunotherapeutic product can include the isolated modified B cell or Blymphocyte cell line that can be a monoclonal B lymphocyte cell line orpolyclonal B lymphocyte cell line that produces one or more secretedimmunoglobulins. The immunotherapeutic product can include the isolatedB lymphocyte cell line that produces one or more secreted antibodies,e.g., antibodies that recognize different epitopes on the same antigen.The immunotherapeutic product can include the isolated B lymphocyte cellline that produces reassigned biological agents (e.g., cytokines,cytotoxins, chemokines, receptors, ligands, immunomodulatory, immuneeffector molecules, transcription factors, etc.). The immunotherapeuticproduct can include the isolated B lymphocyte cell line as one or moreantigen presenting cells.

Thus, the reassigned biological agent can include an agent (such as acytokine or ligand) that is typically not expressed by naturallyoccurring B lymphocytes, or is not expressed under certain circumstancesor conditions by naturally occurring B lymphocytes. Our modified Blymphocytes are able to express the reassigned biological agent due tothe engineering of that B lymphocyte, which may include constitutiveexpression of the reassigned biological agent, or may include inductiveexpression under specific circumstances or conditions (for example,“triggering” of expression by receptor/ligand binding on the surface ofthe modified B lymphocyte).

The isolated B lymphocyte cell line can include an immunotherapeuticproduct administered to a vertebrate subject to develop long-livedisolated B lymphocytes in the vertebrate subject for immune surveillanceof chronic disease. The immunotherapeutic product can include theisolated B lymphocyte cell line having a reassigned biological agent tomodulate immunity for therapy of chronic or acute disease (for example,IL-10 for multiple sclerosis or IL-2 for influenza). Theimmunotherapeutic product can include the isolated B lymphocyte cellline having an endogenously-derived or exogenously derived membraneimmunoglobulin that can be administered to a vertebrate subject toprovide an antigen presenting cell to the vertebrate subject.

In an embodiment, an isolated modified B cell includes at least onereassigned biological agent incorporated at an active Ig gene location(e.g., H or L chain) in a memory B cell. In an embodiment, thereassigned biological agent is under the control of the Igpromoter/enhancer elements and the endogenous antibody (secreted and/ormembrane) of the isolated modified B cell is disrupted. In anembodiment, the isolated modified B cell has an exogenous membrane Ig (Bcell receptor) that binds to an antigen and induces expression of thereassigned biological agent. In an embodiment, the exogenous membrane Ig(B cell receptor) and reassigned biological agent can both be expressedat the same active Ig locus (e.g., H chain).

An isolated cell line as described herein can include an isolated Blymphocyte cell line capable of expressing at least one exogenouslyincorporated membrane immunoglobulin reactive to a first antigen and atleast one endogenous secreted immunoglobulin reactive to a secondantigen. The isolated B lymphocyte cell line is capable of expressing atleast one endogenous membrane immunoglobulin reactive to the secondantigen. The at least one exogenously incorporated membraneimmunoglobulin can include one or more exogenously incorporated membraneimmunoglobulin polypeptides. The at least one exogenously incorporatedmembrane immunoglobulin can include at least one exogenouslyincorporated nucleic acid encoding the at least one membraneimmunoglobulin, wherein the cell line is capable of expressing the atleast one membrane immunoglobulin. The at least one exogenouslyincorporated membrane immunoglobulin comprises at least two exogenouslyincorporated nucleic acid encoding the at least one membraneimmunoglobulin. The at least one exogenously incorporated membraneimmunoglobulin can include nucleic acids encoding two heavy chain (H)immunoglobulins and two light chain (L) immunoglobulins. The at leastone exogenously incorporated membrane immunoglobulin can include nucleicacids encoding one heavy chain (H) immunoglobulin and one light chain(L) immunoglobulin. The at least one exogenously incorporated membraneimmunoglobulin can include nucleic acids encoding one single chain Fv(SCFv) immunoglobulin (e.g., SCFv fused with immunoglobulin constantregion domains). The exogenously incorporated nucleic acid encoding theat least one membrane immunoglobulin can be present in one or morechromosomal loci in the isolated B lymphocyte cell line. The exogenouslyincorporated nucleic acid in the isolated B lymphocyte cell line iscapable of disrupting expression of the endogenous immunoglobulin. Forexample, disruption of endogenous H or L chain expression may knock outproduction of member IgG and/or secreted IgG and more generally mayknock out endogenous antibody synthesis.

In an embodiment, inserting the exogenously incorporated membraneimmunoglobulin, exogenously incorporated secreted immunoglobulin, orexogenously incorporated cytotoxicity effector molecule at an activesite (e.g. using CRISPR technology as described herein) allows for ahijacking of the endogenous machinery (e.g., upon rearrangement of theregions, the promoter and enhancer elements are brought into closeproximity).

The at least two exogenously incorporated nucleic acids encoding the atleast one of the membrane immunoglobulin can be present in Ig H chainand Ig L chain chromosomal loci in the isolated B lymphocyte cell line.The at least one exogenously incorporated nucleic acid encoding the atleast one membrane immunoglobulins can be present in one or more non-IgL chain or non-Ig H chain chromosomal loci in the isolated B lymphocytecell line. The at least one exogenously incorporated nucleic acidencoding the at least one membrane immunoglobulin can be present in anextrachromosomal replicating genetic element in the isolated Blymphocyte cell line. The at least one exogenously incorporated nucleicacid encoding the at least one membrane immunoglobulin can be derivedfrom a B lymphocyte cell line. The at least one exogenously incorporatedmembrane immunoglobulin activated by the first antigen is capable ofcontrolling expression of the at least one endogenous secretedimmunoglobulin reactive to the second antigen. The isolated B lymphocytecell line can include at least one of naïve B lymphocyte, immature Blymphocyte, transitional B lymphocyte, mature B lymphocyte, B1 Blymphocyte, marginal zone B lymphocyte, follicular B lymphocyte, memoryB lymphocyte, plasmablast, or plasma cell. The isolated B lymphocytecell line can include a polyclonal population of B lymphocytes. Theisolated B lymphocyte cell line can include a monoclonal population of Blymphocytes. The membrane immunoglobulin can include at least one of amembrane anchor, a cytoplasmic domain, a hinge region and anextracellular ligand-binding domain.

In an embodiment, the exogenously incorporated nucleic acid encoding theat least one membrane immunoglobulin can be integrated into one or morelocations along the genes encoding the B cell receptor. In anembodiment, the exogenously incorporated nucleic acid encoding the atleast one membrane immunoglobulin can be integrated into one or morelocations in the Ig loci (e.g., the heavy chain or light chainimmunoglobulins as described above).

An isolated recombinant cell line as described herein can include anisolated B lymphocyte cell line capable of expressing at least oneexogenously incorporated membrane immunoglobulin reactive to a firstantigen and at least one exogenously incorporated nucleic acid encodingsecreted immunoglobulin reactive to a second antigen. The isolated Blymphocyte cell line is capable of expressing at least one exogenouslyincorporated nucleic acid encoding membrane immunoglobulin reactive tothe second antigen. The isolated B lymphocyte cell line is capable ofexpressing at least one exogenously incorporated nucleic acid encoding asecreted immunoglobulin reactive to a third antigen. The second antigenand the third antigen can be different epitopes of a single antigenicpolypeptide. The at least one exogenously incorporated membraneimmunoglobulin can include at least one exogenously incorporatedmembrane immunoglobulin polypeptide. The at least one exogenouslyincorporated membrane immunoglobulin can include at least oneexogenously incorporated nucleic acid encoding at least one membraneimmunoglobulin polypeptide, wherein the cell line is capable ofexpressing the at least one membrane immunoglobulin polypeptide. The atleast one exogenously incorporated nucleic acid encoding the at leastone membrane immunoglobulin can be present in one or more chromosomalloci in the isolated B lymphocyte cell line. The at least twoexogenously incorporated nucleic acids encoding the at least onemembrane immunoglobulins can be present in Ig H chain and Ig L chainchromosomal loci in the isolated B lymphocyte cell line. The at leastone exogenously incorporated nucleic acids encoding the at least onemembrane immunoglobulin can be present in one or more non-Ig L or non-IgH chromosomal loci in the isolated B lymphocyte cell line. The at leastone exogenously incorporated nucleic acids encoding the at least onemembrane immunoglobulin can be present in an extrachromosomalreplicating genetic element in the isolated B lymphocyte cell line. Thenucleic acid encoding the at least one membrane immunoglobulin can bederived from a B lymphocyte cell line. The at least one exogenouslyincorporated membrane immunoglobulin activated by the first antigen iscapable of controlling expression of the at least one exogenouslyincorporated secreted immunoglobulin reactive to the second antigen. Theisolated B lymphocyte cell line can include at least one of naïve Blymphocyte, immature B lymphocyte, transitional B lymphocyte, mature Blymphocyte, B1 B lymphocyte, marginal zone B lymphocyte, follicular Blymphocyte, memory B lymphocyte, plasmablast, or plasma cell. Theisolated B lymphocyte cell line can include a polyclonal population of Blymphocytes. The isolated B lymphocyte cell line can include amonoclonal population of B lymphocytes. The membrane immunoglobulin caninclude at least one of a membrane anchor, a cytoplasmic domain, a hingeregion, and an extracellular ligand-binding domain.

In an embodiment, a modified B lymphocyte includes structural orfunctional features for exhibiting cellular cytotoxicity. For example,in an embodiment, a modified B lymphocyte cell or cell line produces oneor more antibodies and has one or more B cell receptors (membraneimmunoglobulins as described herein) that are specific to targetantigens, such as tumor antigens (including but not limited to, antigensthat are mutant forms of “normal” cellular antigens, as well as antigensthat are modified by way of post-translational modifications, andantigens that are expressed in an abnormal way or in an abnormal level).In an embodiment, a modified B lymphocyte cell or cell line is capableof mounting a complete immune response with both humoral as well ascellular immune components. Thus, cytotoxicity expression can includesecreted antibody or antibodies. Cytotoxicity expression can alsoinclude direct cell-to-cell contact that induces death (e.g., by lysis,necrosis, apoptosis, etc.). One of the goals of various embodiments isto provide highly specific, highly effective killing of target cells bythe modified B lymphocytes described herein. In an embodiment, thetarget cells include cancer cells (e.g., tumor or other cancer cells).In an embodiment, the target cells include cells that are related toautoimmune disease or infection. In an embodiment, the target cellsinclude donor or host cells that are reactive to donor cells from acell, tissue, or organ transplant (e.g., graft-versus-host disease). Inan embodiment, the target cells include cells related to pathologicalinflammation or infection.

A method for producing an immunoglobulin in an isolated B lymphocytecell line as described herein can include isolating from a vertebratesubject exposed to, e.g., by infection, or immunized with at least onesecond antigen, a B lymphocyte cell line expressing at least oneendogenous secreted immunoglobulin reactive to the at least one secondantigen; introducing into the isolated B lymphocyte cell line at leastone exogenous membrane immunoglobulin reactive to at least one firstantigen to produce a recombinant B lymphocyte cell line; and selectingthe isolated B lymphocyte cell line expressing the membraneimmunoglobulin reactive to the at least one first antigen and expressingthe at least one endogenous secreted immunoglobulin reactive to the atleast one second antigen. The method of claim can include administeringthe at least one first antigen to stimulate the recombinant B lymphocytecell line; and assessing production of the at least one endogenoussecreted immunoglobulin reactive to the at least one second antigen inthe recombinant B lymphocyte cell line. In the method, introducing intothe at least one isolated recombinant B lymphocyte cell line at leastone exogenous membrane immunoglobulin reactive to the at least one firstantigen can include introducing at least one exogenous membraneimmunoglobulin polypeptide reactive to the at least one first antigen.Introducing into the at least one isolated recombinant B lymphocyte cellline at least one exogenous membrane immunoglobulin reactive to the atleast one first antigen can include introducing at least one exogenousnucleic acid encoding at least one membrane immunoglobulin reactive tothe at least one first antigen. The method can include exposing therecombinant B lymphocyte cell line to the at least one first antigen toactivate the recombinant B lymphocyte cell line to express theendogenous secreted immunoglobulin reactive to the at least one secondantigen. The method can include isolating the endogenous secretedimmunoglobulin reactive to the at least one second antigen from therecombinant B lymphocyte cell line or from a culture of the recombinantB lymphocyte cell line. In the method, activating the at least oneexogenously incorporated membrane immunoglobulin with the first antigenis capable of controlling expression of the at least one exogenouslyincorporated nucleic acid encoding at least one secreted immunoglobulinreactive to the second antigen. The isolated B lymphocyte cell line caninclude at least one of naïve B lymphocytes, immature B lymphocytes,transitional B lymphocytes, mature B lymphocytes, follicular Blymphocytes, memory B lymphocytes, plasmablasts, or plasma cells. Theisolated B lymphocyte cell line can include at least one memory Blymphocyte.

In an embodiment, the isolated B lymphocyte cell line that has beenmodified by way of a chimeric B cell receptor or recombinant B cellreceptor includes utilizing scFv fragments in construction of themodified B cell receptor. In an embodiment, as described herein,transcription factors can also be constructed (e.g., on a separatevector) to be part of the modified B cell line(s).

A method for treating a subject that is afflicted with a disease ordisorder (e.g., an autoimmune disease, cancer, or infection, etc.)includes administering a therapeutically effective amount of an isolatedmodified B lymphocyte cell line as disclosed herein. It is recognizedthat a therapeutically effective amount of cells to be given to asubject can be determined utilizing standard methods for immunotherapyand cell therapy programs.

A method for treating a disease in a vertebrate subject with animmunotherapeutic product as described herein can include isolating froma vertebrate subject exposed to, e.g., by infection, or immunized withat least one second antigen, a B lymphocyte cell line expressing atleast one endogenous secreted immunoglobulin reactive to the at leastone second antigen; introducing into the isolated B lymphocyte cell lineat least one exogenous membrane immunoglobulin reactive to at least onefirst antigen to produce a recombinant B lymphocyte cell line; andselecting the recombinant B lymphocyte cell line expressing the membraneimmunoglobulin reactive to the at least one first antigen and expressingthe at least one endogenous secreted immunoglobulin reactive to the atleast one second antigen for administration to one or more vertebratesubjects. The method can include administering the at least one firstantigen to stimulate the recombinant B lymphocyte cell line; and testingfor the presence of the at least one endogenous secreted immunoglobulinreactive to the at least one second antigen in the recombinant Blymphocyte cell line. The method can include administering to thevertebrate subject a pharmaceutical composition including the isolated Blymphocyte cell line; and administering to the vertebrate subject the atleast one first antigen to stimulate the isolated B lymphocyte cell lineto produce the at least one endogenous secreted immunoglobulin reactiveto the at least one second antigen. The method can include confirmingthe presence of the at least one endogenous secreted immunoglobulinreactive to the at least one second antigen in a bloodstream of thevertebrate subject. The method can include administering the at leastone first antigen to stimulate the recombinant B lymphocyte cell line;testing for the presence of the at least one endogenous secretedimmunoglobulin reactive to the at least one second antigen; andadministering to the vertebrate subject a pharmaceutical compositionincluding the stimulated recombinant B lymphocyte cell line. Therecombinant B lymphocyte cell line can be autologous to one of the oneor more vertebrate subjects. The recombinant B lymphocyte cell line canbe allogeneic to the one or more vertebrate subjects.

A method for producing at least one immunoglobulin in an isolated cellline as described herein can include introducing into at least oneisolated B lymphocyte cell line at least one exogenous membraneimmunoglobulin reactive to at least one first antigen to produce atleast one first isolated B lymphocyte cell line; selecting the at leastone first isolated B lymphocyte cell line expressing the membraneimmunoglobulin reactive to the at least one first antigen; introducinginto the at least one first isolated B lymphocyte cell line at least oneexogenous nucleic acid encoding one or more secreted immunoglobulinsreactive to at least one second antigen to produce at least one isolatedrecombinant B lymphocyte cell line; and selecting the at least oneisolated recombinant B lymphocyte cell line expressing the one or moresecreted immunoglobulin reactive to the at least one second antigen. Themethod can include selecting the at least one isolated recombinant Blymphocyte cell line expressing the at least one exogenous membraneimmunoglobulin reactive to the at least one first antigen. The methodcan include administering the at least one first antigen to stimulatethe at least one isolated recombinant B lymphocyte cell line; andtesting for the presence of the one or more secreted immunoglobulinsreactive to the at least one second antigen in the at least one isolatedrecombinant B lymphocyte cell line. The method can include introducinginto the at least one first isolated B lymphocyte cell line at least oneexogenous membrane immunoglobulin reactive to the at least one secondantigen. The method can include introducing into the at least oneisolated recombinant B lymphocyte cell line at least one exogenousnucleic acid sequence encoding one or more secreted immunoglobulinsreactive to at least one third antigen to produce at least one isolatedsecond recombinant B lymphocyte cell line; and selecting the at leastone isolated second recombinant B lymphocyte cell line expressing the atleast one secreted immunoglobulin reactive to the at least one secondantigen and the at least one secreted immunoglobulin reactive to the atleast one third antigen. The method can include administering the atleast one first antigen to stimulate the at least one isolated secondrecombinant B lymphocyte cell line; and testing for the presence of theat least one exogenous secreted immunoglobulin reactive to the at leastone third antigen in the recombinant B lymphocyte cell line. In themethod, introducing into the at least one isolated B lymphocyte cellline the at least one exogenous membrane immunoglobulin reactive to theat least one first antigen can include introducing at least oneexogenous membrane immunoglobulin reactive to the at least one firstantigen. Introducing into the at least one isolated B lymphocyte cellline the at least one exogenous membrane immunoglobulin reactive to theat least one first antigen can include introducing an exogenous nucleicacid encoding at least one membrane immunoglobulin reactive to the atleast one first antigen. Introducing into the at least one firstisolated B lymphocyte cell line the at least one exogenous membraneimmunoglobulin reactive to the at least one second antigen can includeintroducing at least one exogenous membrane immunoglobulin polypeptidereactive to the at least one second antigen. Introducing into the atleast one first isolated B lymphocyte cell line the at least oneexogenous membrane immunoglobulin reactive to the at least one secondantigen can include introducing at least one exogenous nucleic acidencoding at least one membrane immunoglobulin reactive to the at leastone second antigen. The method can include exposing the at least oneisolated recombinant B lymphocyte cell line to the at least one firstantigen, and testing for the activation of the at least one isolatedrecombinant B lymphocyte cell line to express the exogenous secretedimmunoglobulin reactive to the at least one second antigen. The methodcan include isolating the exogenous secreted immunoglobulin reactive tothe at least one second antigen from the at least one isolatedrecombinant B lymphocyte cell line or from a culture of the at least oneisolated recombinant B lymphocyte cell line. In the method, activatingthe at least one exogenously incorporated membrane immunoglobulin withthe first antigen is capable of controlling expression of the at leastone exogenously incorporated nucleic acid encoding at least one secretedimmunoglobulin reactive to the second antigen. The at least one isolatedB lymphocyte cell line can include at least one of naïve B lymphocytes,immature B lymphocytes, transitional B lymphocytes, mature Blymphocytes, marginal zone B lymphocytes, B1 B lymphocytes, follicular Blymphocytes, memory B lymphocytes, plasmablasts, or plasma cells. The atleast one isolated B lymphocyte cell line can include at least onememory B lymphocyte.

A method for treating a disease in a vertebrate subject with animmunotherapeutic product as described herein can include introducinginto at least one isolated B lymphocyte cell line at least one exogenousmembrane immunoglobulin reactive to at least one first antigen toproduce at least one first isolated B lymphocyte cell line; selectingthe at least one first isolated B lymphocyte cell line expressing themembrane immunoglobulin reactive to the at least one first antigen;introducing into the at least one first isolated B lymphocyte cell lineat least one exogenous nucleic acid encoding one or more secretedimmunoglobulins reactive to at least one second antigen to produce atleast one isolated recombinant B lymphocyte cell line; selecting the atleast one isolated recombinant B lymphocyte cell line expressing thesecreted one or more immunoglobulin reactive to the at least one secondantigens for administration to one or more vertebrate subjects. Themethod can include selecting the at least one isolated recombinant Blymphocyte cell line expressing the at least one exogenous membraneimmunoglobulin reactive to the at least one first antigen. The methodcan include administering the at least one first antigen to stimulatethe at least one isolated recombinant B lymphocyte cell line; andtesting for the presence of the one or more secreted immunoglobulinreactive to the at least one second antigen in the at least one isolatedrecombinant B lymphocyte cell line. The method can include administeringto the vertebrate subject a pharmaceutical composition including the atleast one isolated recombinant B lymphocyte cell line; and administeringto the vertebrate subject the at least one first antigen to stimulatethe at least one isolated recombinant B lymphocyte cell line to producethe one or more exogenous secreted immunoglobulin reactive to the atleast one second antigen. The method can include confirming the presenceof the at least one exogenous secreted immunoglobulin reactive to the atleast one second antigen in a bloodstream of the vertebrate subject. Themethod can include administering the at least one first antigen tostimulate the at least one isolated recombinant B lymphocyte cell lineto produce the one or more exogenous secreted immunoglobulin reactive tothe at least one second antigen; and administering to the vertebratesubject a pharmaceutical composition including the stimulated at leastone isolated recombinant B lymphocyte cell line. The method can includeintroducing into the at least one first isolated B lymphocyte cell lineat least one exogenous membrane immunoglobulin reactive to the at leastone second antigen. The method can include introducing into the at leastone isolated recombinant B lymphocyte cell line at least one exogenousnucleic acid encoding one or more secreted immunoglobulins reactive toat least one third antigen to produce at least one isolated secondrecombinant B lymphocyte cell line; and selecting the at least oneisolated second recombinant B lymphocyte cell line expressing at leastone of the secreted immunoglobulin reactive to the at least one secondantigen and the secreted immunoglobulin reactive to the at least onethird antigen. The method can include administering to the vertebratesubject a pharmaceutical composition including the at least one isolatedsecond recombinant B lymphocyte cell line; and administering to thevertebrate subject the at least one first antigen to stimulate the atleast one isolated second recombinant B lymphocyte cell line to producethe one or more exogenous secreted immunoglobulin reactive to the atleast one second antigen and the one or more exogenous secretedimmunoglobulin reactive to the at least one third antigen. The methodcan include confirming the presence of the at least one exogenoussecreted immunoglobulin reactive to the at least one second antigen andthe one or more exogenous secreted immunoglobulin reactive to the atleast one third antigen in a bloodstream of the vertebrate subject. Themethod can include administering to the vertebrate subject the at leastone first antigen to stimulate the at least one isolated secondrecombinant B lymphocyte cell line to produce the one or more exogenoussecreted immunoglobulin reactive to the at least one second antigen andthe one or more exogenous secreted immunoglobulin reactive to the atleast one third antigen; and administering to the vertebrate subject apharmaceutical composition including the stimulated at least oneisolated second recombinant B lymphocyte cell line. The recombinant Blymphocyte cell line can be autologous to one of the one or morevertebrate subjects. The recombinant B lymphocyte cell line can beallogeneic to the one or more vertebrate subjects.

A method for producing at least one immunoglobulin in an isolated cellline as described herein can include introducing into at least one firstisolated B lymphocyte cell line at least one exogenous nucleic acidencoding one or more secreted immunoglobulins reactive to at least onefirst antigen to produce at least one isolated recombinant B lymphocytecell line; selecting the at least one isolated recombinant B lymphocytecell line expressing the one or more secreted immunoglobulin reactive tothe at least one first antigen; introducing into the at least oneisolated B lymphocyte cell line at least one exogenous membraneimmunoglobulin reactive to at least one second antigen to produce atleast one first isolated B lymphocyte cell line; and selecting the atleast one first isolated B lymphocyte cell line expressing the membraneimmunoglobulin reactive to the at least one second antigen.

A method for treating a disease in a vertebrate subject with animmunotherapeutic product as described herein can include introducinginto at least one first isolated B lymphocyte cell line at least oneexogenous nucleic acid encoding one or more secreted immunoglobulinsreactive to at least one first antigen to produce at least one isolatedrecombinant B lymphocyte cell line; selecting the at least one isolatedrecombinant B lymphocyte cell line expressing the secreted one or moreimmunoglobulin reactive to the at least one first antigens; introducinginto the at least one isolated B lymphocyte cell line at least oneexogenous membrane immunoglobulin reactive to at least one secondantigen to produce at least one first isolated B lymphocyte cell line;and selecting the at least one first isolated B lymphocyte cell lineexpressing the membrane immunoglobulin reactive to the at least onesecond antigen for administration to the vertebrate subject.

The foregoing summary is illustrative only and is not intended to be inany way limiting. In addition to the illustrative aspects, embodiments,and features described above, further aspects, embodiments, and featureswill become apparent by reference to the drawings and the followingdetailed description.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a schematic of a diagrammatic view of hypotheticalimmunoglobulin genes for memory B lymphocytes.

FIGS. 2A, 2B, 2C are a schematic of a diagrammatic view of nonfunctionaland functional immunoglobulin heavy chain genes on chromosomes 14.

FIGS. 3A, 3B, 3C are a schematic of a diagrammatic view of replacementat immunoglobulin loci with heavy chain genes to express membrane IgGand secreted IgG.

FIGS. 4A, 4B, 4C, 4D are a schematic of a diagrammatic view of protocolsto produce recombinant B lymphocytes with membrane immunoglobulin to afirst antigen and secreted immunoglobulin to a second antigen.

FIG. 5 is a schematic of a diagrammatic view of a method for producingan immunoglobulin in an isolated B lymphocyte cell line.

FIG. 6 is a schematic of a diagrammatic view of a method for producingan immunoglobulin in an isolated B lymphocyte cell line.

FIG. 7 is a schematic of a diagrammatic view of a method for producingan immunoglobulin in an isolated B lymphocyte cell line.

FIG. 8A is a schematic of a diagrammatic view of a recombinant B cellreceptor protein.

FIG. 8B is a schematic of a diagrammatic view of a recombinant B cellreceptor expression vector.

FIG. 8C is a schematic of a diagrammatic view of chromosome 14 withinserted gene.

FIG. 8D is a schematic of a diagrammatic view of an expression vectorwith transcription factors.

FIG. 9A is a schematic of a diagrammatic view of an example ofintegration of a desired expression construct at an endogenous site(light chain Ig).

FIG. 9B is a schematic of a diagrammatic view of an example ofintegration of a desired expression construct at an endogenous site(heavy chain Ig).

FIG. 10 is a schematic of a diagrammatic view of a modified B cellengineered to selectively engage a surface immunoglobulin with a firsttarget antigen and subsequently secrete a predetermined antibody to asecond target antigen with optional secretion of a reassigned biologicalagent and/or cytotoxic effector molecule(s).

FIG. 11 is a schematic of a diagrammatic view of a lymph node withmodified B cells having reactivity to selective antigens, as determinedby the particular modification(s) of the B cells.

FIG. 12A is a schematic of a diagrammatic view of an example ofintegration of a desired expression construct at an endogenous site(Heavy Chain Ig).

FIG. 12B is a schematic of a diagrammatic view of an example of anexpression vector for use in modifying B cells as described herein.

FIG. 13 is a schematic of a diagrammatic view of an example of anintegration of a desired bicistronic expression construct at anendogenous site (Heavy Chain Ig).

DETAILED DESCRIPTION

In the following detailed description, reference is made to theaccompanying drawings, which form a part hereof. In the drawings,similar symbols typically identify similar components, unless contextdictates otherwise. The illustrative embodiments described in thedetailed description, drawings, and claims are not meant to be limiting.Other embodiments may be utilized, and other changes may be made,without departing from the spirit or scope of the subject matterpresented here.

Compositions and methods are disclosed herein for producing one or moreimmunoglobulins in an isolated B lymphocyte cell line. Compositions andmethods are disclosed herein for producing one or more immunoglobulinsin the isolated B lymphocyte cell line that direct cell signaling bymembrane immunoglobulin in the isolated B lymphocyte cell line. Immunecell therapy in a vertebrate subject can include administering to thevertebrate subject the isolated B lymphocyte cell line that synthesizessecreted immunoglobulins and membrane immunoglobulins each havingdifferent target antigens. Immune cell therapy in a vertebrate subjectcan include administering to the vertebrate subject antigen presentingcells comprised of the isolated B lymphocyte cell line that directsantigen internalization and processing to produce exceptional antigenpresenting cells. The isolated B lymphocyte cell line can produceantigen presenting cells that are exceptional or superior at capturing,internalizing and presenting the antigen recognized by the endogenous orexogenously derived membrane immunoglobulin. Compositions and methodsare disclosed herein for treating a disease in a vertebrate subject withan immunotherapeutic product. The immunotherapeutic product can includethe isolated B lymphocyte cell line having an endogenously-derived orexogenously derived membrane immunoglobulin reactive to a first antigenwherein the isolated B lymphocyte cell line produces one or moresecreted immunoglobulins reactive to a second antigen. Theimmunotherapeutic product can include the isolated B lymphocyte cellline that can be a monoclonal B lymphocyte cell line or polyclonal Blymphocyte cell line that produces one or more secreted antibodiesand/or a reassigned biological agent. The immunotherapeutic product caninclude the isolated B lymphocyte cell line that produces one or moresecreted antibodies, e.g., antibodies that recognize different epitopeson the same antigen. The immunotherapeutic product can include theisolated B lymphocyte cell line as one or more antigen presenting cells.

The isolated B lymphocyte cell line can include an immunotherapeuticproduct administered to a vertebrate subject to develop long-livedisolated B lymphocytes in the vertebrate subject for immune surveillanceof chronic disease. The immunotherapeutic product can include theisolated B lymphocyte cell line having an endogenously-derived orexogenously derived membrane immunoglobulin that can be administered toa vertebrate subject to provide an antigen presenting cell to thevertebrate subject.

An isolated cell line as described herein can include an isolated Blymphocyte cell line capable of expressing at least one exogenouslyincorporated membrane immunoglobulin reactive to a first antigen and atleast one endogenous secreted immunoglobulin reactive to a secondantigen. The at least one exogenously incorporated membraneimmunoglobulin can include an exogenously incorporated membraneimmunoglobulin polypeptide. The at least one exogenously incorporatedmembrane immunoglobulin can include an exogenously incorporated nucleicacid encoding a membrane immunoglobulin polypeptide, wherein the cellline is capable of expressing the membrane immunoglobulin polypeptide.

An isolated recombinant cell line as described herein can include anisolated B lymphocyte cell line capable of expressing at least oneexogenously incorporated membrane immunoglobulin reactive to a firstantigen and at least one exogenously incorporated nucleic acid encodingsecreted immunoglobulin reactive to a second antigen.

An isolated recombinant cell line as described herein can include anisolated B lymphocyte cell line capable of expressing at least oneexogenously incorporated gene integrated at an active, rearrangedimmunoglobulin gene under the control of immunoglobulin variable regionpromoters and immunoglobulin enhancers. For example, expression of agene for a reassigned biological agent integrated at a rearrangedimmunoglobulin H-chain gene under the control of a variable heavy chainpromoter and the immunoglobulin mu enhancer.

An isolated recombinant B cell line as described herein can include thecapability of expressing a reassigned biological agent, such as aprotein, glycoprotein, proteoglycan, nucleic acid (RNA, DNA, PNA, etc.),or other biological agent that is not ordinarily expressed from the Igchromosomal loci for Ig H-chain and Ig L-chain in a naturally occurringB cell. For example, the recombinant B cells described herein caninclude the capability of expressing at least one cytokine, cytokinereceptor, small molecule, protein, monosaccharide, disaccharide,polysaccharide, or other biological agent. In an embodiment, thereassigned biological agent can include at least one enzyme,G-protein-coupled receptor, or ligand. In an embodiment, the reassignedbiological agent can include at least one of tumor necrosis factor(TNF), TNF-related apoptosis-inducing ligand (TRAIL/Apo2L), OX-40, CD95(FasL/Apo-1L), gamma interferon (γ-IFN), perforin, interleukin-21(IL-21), IL-12, IL-15, IL-10, IL-22, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7,IL-12, IL-15, IL-17, IL-18, IL-23, pathogen-associated molecularpatterns (PAMPs), damage-associated molecular patterns (DAMPs), CXCL-1,CXC, CC, GM-CSF, G-CSF, M-CSF, stem cell factor, TGF-beta, INFgamma,INFalpha, TNFalpha, or other cytokine. In an embodiment, the reassignedbiological agent can include at least one of a tumor-associated antigen,cell surface antigen or viral antigen. For example, tumor associatedantigens can include: MUC-1, MUC-16, prostate cancer membrane antigen(PCMA), epidermal growth factor receptor 2 (HER2), B cell maturationantigen (BCMA), CD38, CD30, MAGE A1, NY-ESO-1 and CD44v6. Viral antigenscan include: HIV-1 proteins: gag, env, gp41, gp120, RNA-dependent DNApolymerase; influenza hemagglutinin; and hepatitis C virus proteins:NS3, NS4 and NS5. In an embodiment, the reassigned biological agentincludes a bacterial component configured to induce other B cells tobecome IgA producing plasma cells. In an embodiment, the isolatedmodified B cell is utilized in the gut of a subject to workcooperatively or competitively with the microbiome in order to maintainhealth or alter a disease state in the subject. See e.g., Shi et al.,Mil. Med. Res. 2017; 4:14, online Apr. 27, 2017, which is incorporatedherein by reference. In an embodiment, the disease state includesdisease that affects the gut. In an embodiment, the disease stateincludes disease that affects another system of the subject's body,including a system disease.

In an embodiment, expression of an agent can be initiated by engagementof the modified B cell's immunoglobulin receptor (BCR). For example,transcription of the reassigned biological agent is initiated in thecascade of events following appropriate antigen binding occurring withthe modified B cell's BCR as described. Antigen binding to membrane IgGleads to tyrosine phosphorylation on Igα and Igβ (signal transductionproteins comprising the BCR) which initiate signaling pathways thatactivate and translocate intracellular messengers and transcriptionfactors (e.g., NF-AT, Ras/Erk, Bright and BTK) which lead to memory Bcell activation and Ig gene expression. (See e.g., Wienands et al.,Current Topics in Microbiology and Immunology 393: 107-121, 2016 andSchmidt et al., EMBO J. 28: 711-724, 2009 which are incorporated hereinby reference.)

In an embodiment, the modified B lymphocyte is modified to express atleast one reassigned biological agent without further modification. Inan embodiment, the modified B lymphocyte is modified to constitutivelyexpress the at least one reassigned biological agent. As describedherein, the reassigned biological agent includes secreted molecules(e.g., cytokines, chemokines, cytotoxins, etc.) and can play a role in alarger immunological response (e.g. stimulating T cells, NK cells,macrophages, epithelial cells, other B cells, neutrophils, basophils,eosinophils, etc.). In an embodiment, the modified B lymphocyte ismodified to inductively express the at least one reassigned biologicalagent (e.g., expression of the at least one reassigned biological agentcan be driven by receptor-ligand binding, by transcription factor, byprotein production of another biological reaction, etc.). In anembodiment, the modified B lymphocyte is also modified in other ways asdescribed herein (e.g., to express an exogenous membrane immunoglobulinreceptor and/or to express an exogenous secreted immunoglobulin receptorand/or to express cytotoxic agents, etc.).

In an embodiment, as described herein, the reassigned biological agentcan include an agent that a naturally occurring B lymphocyte would notordinarily express at all but due to its modification, the modified Blymphocyte is capable of expressing the reassigned biological agent. Inan embodiment, as described herein, the reassigned biological agent caninclude an agent that a naturally occurring B lymphocyte would notordinarily express in specific circumstances or conditions but due toits modification, the modified B lymphocyte is capable of expressing thereassigned biological agent under those specific circumstances orconditions. For example, under certain circumstances, a naturallyoccurring regulatory B lymphocyte expresses and secretes IL-10 orTFG-beta 1, while a naturally occurring effector B lymphocyte producescytokines such as IL-2, IL-4, TNFalpha, IL-6, or INFgamma. Thedetermination of a regulatory B lymphocyte or effector B lymphocyte isbased on the exposure of those particular cells to antigen and/or othercytokines and immune modulators. (See, e.g., Lund, Curr. Opin. Immunol.2008 June; 20(3):332-338, which is incorporated by reference herein.)

With regard to our modified B lymphocytes, a B lymphocyte that would(under naturally occurring circumstances) express IL-2, can instead bemodified to secrete IL-10, for example. Thus, the IL-10 would fulfillthe role of a reassigned biological agent. Conversely, if a naturallyoccurring B lymphocyte would (under naturally occurring circumstances)express IL-10, but it is modified to constitutively or inductivelysecrete IL-2 regardless of the particular immunological conditions, thenIL-2 would fulfill the role of a reassigned biological agent. Thus, themodification of a reassigned biological agent acts as a powerful tool todirect immune responses both in the modified B lymphocytes themselves,as well as the other players in the immune reaction (epithelial cells,neurons, other immune cells, etc.). This can be particularly useful, forexample, with regard to “misplaced” immune reactions such as with tumorimmunology (e.g., tumor suppression of standard immune response to tumorantigens), infectious disease (e.g., viral evasion of standard immunesurveillance) or autoimmunity (e.g., heightened inflammation or highlyreactive immune response to “self” or “no danger” antigens), etc.

A method for producing an immunoglobulin in an isolated B lymphocytecell line as described herein can include isolating from a vertebratesubject exposed to, e.g., by infection, or immunized with at least onesecond antigen, a B lymphocyte cell line expressing at least oneendogenous secreted immunoglobulin reactive to the at least one secondantigen; introducing into the isolated B lymphocyte cell line at leastone exogenous membrane immunoglobulin reactive to at least one firstantigen to produce a recombinant B lymphocyte cell line; and selectingthe isolated B lymphocyte cell line expressing the membraneimmunoglobulin reactive to the at least one first antigen and expressingthe at least one endogenous secreted immunoglobulin reactive to the atleast one second antigen. As described herein, the modification

A method for treating a disease in a vertebrate subject with animmunotherapeutic product as described herein can include isolating froma vertebrate subject exposed to, e.g., by infection, or immunized withat least one second antigen, a B lymphocyte cell used to generate a cellline expressing at least one endogenous secreted immunoglobulin reactiveto the at least one second antigen; introducing into the isolated Blymphocyte cell at least one exogenous membrane immunoglobulin reactiveto at least one first antigen to produce a recombinant B lymphocyte cellline; and selecting the recombinant B lymphocyte cell line expressingthe membrane immunoglobulin reactive to the at least one first antigenand expressing the at least one endogenous secreted immunoglobulinreactive to the at least one second antigen for administration to one ormore vertebrate subjects.

A method for producing at least one immunoglobulin in an isolated cellline as described herein can include introducing into at least oneisolated B lymphocyte cell at least one exogenous membraneimmunoglobulin reactive to at least one first antigen to produce atleast one first isolated B lymphocyte cell line; selecting the at leastone first isolated B lymphocyte cell line expressing the membraneimmunoglobulin reactive to the at least one first antigen; introducinginto the at least one first isolated B lymphocyte cell line at least oneexogenous nucleic acid encoding one or more secreted immunoglobulinsreactive to at least one second antigen to produce at least one isolatedrecombinant B lymphocyte cell line; and selecting the at least oneisolated recombinant B lymphocyte cell line expressing the one or moresecreted immunoglobulin reactive to the at least one second antigen.

A method for treating a disease in a vertebrate subject with animmunotherapeutic product as described herein can include introducinginto at least one isolated B lymphocyte cell at least one exogenousmembrane immunoglobulin reactive to at least one first antigen toproduce at least one first isolated B lymphocyte cell line; selectingthe at least one first isolated B lymphocyte cell line expressing themembrane immunoglobulin reactive to the at least one first antigen;introducing into the at least one first isolated B lymphocyte cell lineat least one exogenous nucleic acid encoding one or more secretedimmunoglobulins reactive to at least one second antigen to produce atleast one isolated recombinant B lymphocyte cell line; selecting the atleast one isolated recombinant B lymphocyte cell line expressing thesecreted one or more immunoglobulin reactive to the at least one secondantigens for administration to one or more vertebrate subjects.

A method for producing at least one immunoglobulin in an isolated cellline as described herein can include introducing into at least one firstisolated B lymphocyte cell at least one exogenous nucleic acid encodingone or more secreted immunoglobulins reactive to at least one firstantigen to produce at least one isolated recombinant B lymphocyte cellline; selecting the at least one isolated recombinant B lymphocyte cellline expressing the one or more secreted immunoglobulin reactive to theat least one first antigen; introducing into the at least one isolated Blymphocyte cell line at least one exogenous membrane immunoglobulinreactive to at least one second antigen to produce at least one firstisolated B lymphocyte cell line; and selecting the at least one firstisolated B lymphocyte cell line expressing the membrane immunoglobulinreactive to the at least one second antigen.

A method for treating a disease in a vertebrate subject with animmunotherapeutic product as described herein can include introducinginto at least one first isolated B lymphocyte cell at least oneexogenous nucleic acid encoding one or more secreted immunoglobulinsreactive to at least one first antigen to produce at least one isolatedrecombinant B lymphocyte cell line; selecting the at least one isolatedrecombinant B lymphocyte cell line expressing the secreted one or moreimmunoglobulin reactive to the at least one first antigens; introducinginto the at least one isolated B lymphocyte cell line at least oneexogenous membrane immunoglobulin reactive to at least one secondantigen to produce at least one first isolated B lymphocyte cell line;and selecting the at least one first isolated B lymphocyte cell lineexpressing the membrane immunoglobulin reactive to the at least onesecond antigen for administration to the vertebrate subject.

An isolated recombinant cell line includes an isolated B lymphocyte cellline capable of expressing at least one endogenous membraneimmunoglobulin reactive to a first antigen and at least one exogenouslyincorporated nucleic acid encoding at least one secreted immunoglobulinreactive to a second antigen.

In an embodiment, a modified B lymphocyte includes structural orfunctional features for exhibiting cellular cytotoxicity. For example,in an embodiment, a modified B lymphocyte cell or cell line produces oneor more antibodies and has one or more B cell receptors (membraneimmunoglobulins as described herein) that are specific to targetantigens, such as tumor antigens (including but not limited to, antigensthat are mutant forms of “normal” cellular antigens, as well as antigensthat are modified by way of post-translational modifications, andantigens that are expressed in an abnormal way or in an abnormal level).In an embodiment, a modified B lymphocyte cell or cell line is capableof mounting a complete immune response with both humoral as well ascellular immune components.

In an embodiment, a modified B lymphocyte that exhibits cytotoxicity iscompetent to express (directly or indirectly) at least one of perforin,granzymes, and other cytotoxic components. For example, in anembodiment, the B cell receptor (membrane bound immunoglobulin) signalsto elicit cytotoxic effectors when engaged with a tumor cell (i.e. the Bcell receptor engages with an antigen of a tumor cell). In anembodiment, the modified lymphocyte is competent to secrete an antibodythat is cytotoxic (e.g., by way of fixing complement or engaging ADCC[antibody-dependent cell-mediated cytotoxicity]) for the same tumorcell(s).

In an embodiment, a modified B lymphocyte cell is derived from B cellsfollowing vaccination with tumor antigens, for example, or from donorperipheral blood lymphocytes by modification of expression of at leastone of antibody or B cell receptor (e.g., chimeric B cell receptor orrecombinant B cell receptor).

In an embodiment, a modified B lymphocyte cell is modified to expresscytotoxicity by way of expression of a recombinant B cell receptor or achimeric receptor with scFv and membrane immunoglobulin forextracellular transmembrane and cytoplasmic domains along with acytoplasmic domain from IL21 receptor and TLR or another signalingmolecule to elicit expression of granzyme, perforin, etc. from themodified B lymphocyte cell.

For example, in the case of HIV infection, the modified B lymphocytecell is able to mount a humoral as well as cytotoxic immune reaction.For example, the modified B lymphocyte cell can secrete neutralizingantibody for HIV particles or virally-infected cells. Optionally, inaddition to the neutralizing antibody, the modified B lymphocyte cellcan directly induce apopotosis or otherwise directly kill HIV infectedcells (e.g. infected T cells in the lymph nodes, known as “reservoirs”of infected T cells not destroyed under current HIV anti-viraltherapies.

Likewise, the modified B lymphocyte cell can target auto-immune cells(e.g. multiple sclerosis cells, arthritis cells, etc.) that can beidentified as self-reactive. In an embodiment, the modified B lymphocytecell induces apoptosis or otherwise directly kills such self-reactivecells. In an embodiment, these self-reactive cells include at least oneof B cells, T cells, macrophages, or other immune cells. In anembodiment, these self-reactive cells include inflammatory cells

In an embodiment, a modified B lymphocyte cell is modified to express arecombinant B cell receptor or chimeric B cell receptor specific for afirst antigen and an antibody recognizing a second antigen, providingincreased specificity as well as increased cellular cytotoxicity andantibody-mediated killing of the target cells (e.g. tumor cells,auto-immune cells, infected cells, inflammatory cells, necrotic cells,regulatory cells (e.g., regulatory T cells, regulatory B cells andmyeloid-derived suppressor cells).

In an embodiment, a modified B lymphocyte cell with chimeric B cellreceptor or recombinant B cell receptor has been modified tospecifically react to one or more target cells, and exhibit cytotoxicityfor the one or more target cells. In an embodiment, the modified Blymphocyte cell or cell line is engineered specifically for reactionwith one or more tumor cells or tumor cell types. In an embodiment, themodified B lymphocyte cell or cell line is engineered through laboratorytechniques and optionally through use of computer data and/or modelingof various components of the B lymphocytes.

In an embodiment, the modified B lymphocyte cell with a chimeric B cellreceptor or recombinant B cell receptor includes a modified receptorthat is competent to transduce signals that induce expression ofcytotoxic effector molecules, when the receptor is engaged.

In an embodiment, the recombinant B cell receptor or chimeric B cellreceptor includes a heterologous extracellular, trans-membrane andcytoplasmic signaling domain(s) that elicit expression of cytotoxiceffector molecules.

In an embodiment, the recombinant B cell receptor or chimeric B cellreceptor includes a cytoplasmic domain derived from at least one of thecommon gamma chain, IL-21R, a Toll-like receptor (TLR) or CD40. In anembodiment, the recombinant B cell receptor or chimeric B cell receptoris competent to elicit expression of cytotoxic effector molecules, suchas perforin, granzyme B, Fas ligand, TRAIL, or others. In an embodiment,the recombinant B cell receptor or chimeric B cell receptor is competentto elicit expression of a TNF family receptor in the target cell, suchas TNFR1, Fas receptor, DR4, DR5, or other “death domain” receptor.

In an embodiment, the modified B cell secretes or expresses at least oneof TNF-alpha ligand, lymphotoxin alpha or beta, OX40L, CD154, LIGHT,TL1-A, CD70, Siva, CD153, 4-1BB, RANKL, TWEAK, APRIL, BAFF, CAMLG, NGF,BDNF, NT-3, NT-4, GITR, TL1A, or EDA-A2.

For example, optimal stimulation for effective expression of CD70 onIFN-alpha-induced monocyte-derived dendritic cells, widely used fortumor immunotherapy, has been studied by exposure to variousmaturation-inducing factors (Toll-like receptor ligands, CD40 ligand andpro-inflammatory mediators, including prostaglandin E2). SeeArimoto-Miyamoto et al., Immunol, 2010 May 130(1): 137-149, which isincorporated herein by reference. Further, the CD70-CD27 interactiondiminished production of IL-10. Id.

For example, expression of CD153 on B cells in the presence of B cellreceptor engagement and IL-4, results in Ig class switching, while CD154expression contributes to CD153 expression. See Cerutti, et al., JImmunol 2000 Jul. 15; 165(2):786-794, which is incorporated herein byreference. The CD153 “switch” for expression of alternate or additionalgenes or expression cassettes can be built into the modified B cellsdescribed herein.

For example, OX40-OX40L interactions have been found to play a role inthe development of several different inflammatory and autoimmunediseases, and which may be targeted for intervention. See for example,Croft, et al., Immunol Rev 2009 May, 229(1): 173-191, which isincorporated herein by reference.

In an embodiment, the recombinant B cell receptor or chimeric B cellreceptor includes at least one extracellular domain specific for one ormore target antigens. In an embodiment, the recombinant B cell receptoror chimeric B cell receptor includes a modified receptor that recognizesa first antigen, and secretes an antibody that recognizes a secondantigen. In an embodiment, the recombinant B cell receptor or chimeric Bcell receptor includes a modified receptor that recognizes a firstepitope of one antigen and secretes an antibody that recognizes a secondepitope of the same antigen. Thus, the modified B cells (withrecombinant B cell receptor or chimeric B cell receptor) can be designedand engineered by laboratory techniques to be responsive to tumor cellswith greater specificity and greater cytotoxic activity.

In an embodiment, the modified B cells that exhibit cytotoxicity arefurther included in embodiments of B cells described herein that exhibita chimeric B cell receptor or recombinant B cell receptor specific forone antigen and secrete antibodies specific for a different antigen thanits B cell receptor. Such memory B cells can also include the targetedcytotoxicity characteristics as described herein.

In an embodiment, the target cells include cells that have been infectedby virus, mycoplasma, bacteria, yeast, or other microorganism. In anembodiment, the target cells include tumor cells, such as primary tumorcells, circulating tumor cells, or metastatic tumor cells. In anembodiment, the target cells include auto-immune cells.

In an embodiment, a method of making the modified B lymphocyte cellsincludes engineering cells by way of laboratory techniques. In anembodiment, a method of using the modified B lymphocytes for treatmentof disease includes administering a therapeutically effective amount ofthe modified B lymphocyte cells to a subject. Specific examples ofmethods of making the modified B lymphocyte cells are described ingreater detail in the Prophetic Examples section herein.

In an embodiment, a modified B lymphocyte cell or cell line as describedherein is engineered, and utilized for administration to a subject fortreatment. As described herein, in an embodiment, the subject has activedisease. In an embodiment, the subject has chronic disease. In anembodiment, the subject has been exposed to a disease-causing agent andmay or may not yet have symptoms of disease. In an embodiment, thesubject has latent disease.

A method for producing an immunoglobulin in a recombinant B lymphocytecell line includes isolating from a vertebrate subject exposed to, e.g.,by infection, or immunized with at least one first antigen, a Blymphocyte cell line expressing at least one endogenous membraneimmunoglobulin reactive to the at least one first antigen; introducinginto the isolated B lymphocyte cell line at least one exogenous nucleicacid encoding at least one of a secreted immunoglobulin reactive to atleast one second antigen to produce a recombinant B lymphocyte cellline; and assaying for presence of the at least one exogenous secretedimmunoglobulin reactive to the at least one second antigen to select therecombinant B lymphocyte cell line.

A method for treating a disease in a vertebrate subject with animmunotherapeutic product includes isolating from a vertebrate subjectexposed to, e.g., by infection, or immunized with at least one firstantigen, a B lymphocyte cell line expressing at least one endogenousmembrane immunoglobulin reactive to the at least one first antigen;introducing into the isolated B lymphocyte cell line at least one of atleast one exogenous nucleic acid encoding at least one secretedimmunoglobulin reactive to at least one second antigen; assaying forpresence of at least one exogenous secreted immunoglobulin reactive tothe at least one second antigen to select the recombinant B lymphocytecell line for administration to the vertebrate subject.

The isolated B lymphocytes can be used for immunotherapy:

-   -   Long-lived isolated B lymphocytes can be used for immune        surveillance of chronic disease.    -   Isolated B lymphocytes having membrane immunoglobulin        recognizing antigen can act as exceptional antigen presenting        cells to present antigen to T lymphocytes.    -   Immunotherapy with polyclonal autologous isolated B lymphocytes        is a valuable protocol. For example, influenza immune B        lymphocytes can be transfected en masse with retroviral vectors.        Alternatively, one may immunize with a vaccine and transfect        multiple isolated B lymphocytes, e.g., polyclonal B lymphocytes,        recognizing different epitopes of the same antigen.

A number of protocols, as presented herein, may be utilized to producean isolated B lymphocyte cell line as stated in more detail in thedetailed description and examples. An isolated B lymphocyte cell linecapable of expressing at least one endogenous membrane immunoglobulinreactive to a first antigen or capable of expressing at least oneendogenous secreted immunoglobulin reactive to a first antigen can bedeveloped by immunizing an individual with a model antigen, e.g.,dinitrophenol (DNP) or an influenza antigen, to elicit memory B cellswith endogenous membrane immunoglobulin, e.g., B cell receptors (BCR),reactive to the DNP model antigen or the influenza antigen and/orendogenous soluble immunoglobulin, e.g., antibody reactive to the DNPantigen or the influenza antigen.

An isolated B lymphocyte cell line capable of expressing at least oneexogenous secreted immunoglobulin reactive to a broadly neutralizinginfluenza antigen can be developed by isolating human B cells from anindividual who is immune to influenza virus infection and immortalizingthe human B cells by infecting the isolated B cells with Epstein Barrvirus (EBV). Methods to clone immunoglobulin heavy (H) chain and light(L) chain genes from the EBV-immortalized B lymphocyte cell line may beused. See e.g., U.S. Pat. No. 7,741,077 issued to Grawunder et al. onJun. 22, 2010 and Early et al., Proc. Natl. Acad. Sci. USA 76: 857-861,1979, which are incorporated herein by reference. To promote homologousrecombination the immunoglobulin genes encoding the H chain and L chainfor a secreted anti-influenza antibody are cloned in plasmid targetingvectors to obtain targeted integration in the correspondingnonfunctional, germline Ig loci on chromosomes 14 and 2 respectively.Alternatively memory B cells obtained from a patient with a chronicviral infection can be genetically engineered by replacing theirfunctional, expressed Ig genes with exogenous Ig genes encoding amembrane immunoglobulin, e.g., anti-DNP antibody. The Ig H and Ig Lchain genes encoding the anti-DNP antibody may be inserted in thefunctional, expressed Ig gene loci on chromosomes 14 and 2 by usingmethods of homologous recombination. See e.g., U.S. Pat. Nos. 5,202,238,6,570,061, and 6,841,383.

Memory B cells expressing anti-DNP membrane IgG can be engineered toexpress Ig genes encoding a secreted IgG antibody specific forinfluenza. The anti-influenza IgG₁ H chain gene (i.e., γ₁-H chain gene)may be engineered to remove coding sequences for the membrane spanningdomain (TM), the cytoplasmic amino acids (Cyt), and a polyA additionsite to yield a γ₁-H chain gene encoding a secreted H chain only.

To obtain human immunoglobulin (Ig) genes encoding a specific antibodyagainst cancer, e.g., PSA, or for infectious disease, a hybridoma cellline that produces the anti-PSA antibody is constructed. For exampletransgenic mice with human Ig genes (e.g., XenoMouse® available fromAbgenix Inc., Fremont, Calif.) are immunized with PSA and their B cellsare fused with a myeloma cell fusion partner, e.g. SP2/0 cells(available from American Type Culture Collection, Manassas, Va.) tocreate hybridoma cell clones expressing human antibodies (see e.g., U.S.Pat. No. 8,013,128 Ibid.). Supernatants from the hybrid clones arescreened using an immunoassay to detect human IgG antibodies which bindPSA protein. Hybridoma clones producing antibodies that recognize PSAare expanded and antibodies from each clone are tested using a Biacore™A100 instrument (available from GE Healthcare, Piscataway, N.J.) tomeasure antibody affinity and specificity for PSA (see e.g., GEHealthcare, Application Note 84, “Early kinetic screening of hybridomas. . . ” which is incorporated herein by reference). Hybridomasexpressing high affinity antibodies for PSA are selected for cloning oftheir human Ig genes, for example, by homologous recombination.

The engineered immunoglobulin genes encoding a membrane immunoglobulinare expressed in a mammalian cell line and the membrane IgG is purifiedfrom the cell line. For example, a kappa (κ) L chain gene and themodified γ-1 H chain gene are inserted in a lentiviral expression vectorusing standard recombinant DNA methods (see e.g., U.S. PatentPublication No. 2007/0116690 by Yang et al. published on May 24, 2007which is incorporated herein by reference). The viral vector is used totransfect Chinese Hamster Ovary (CHO) cells (available from AmericanType Culture Collection, Manassas, Va.) which are engineered to expressthe membrane immunoglobulin.

To insure that the recombinant memory B cells are safe for use inpatients a suicide gene may be introduced into the B cells. To stopuncontrolled proliferation (and other adverse events) by the recombinantmemory B cells, a suicide gene, herpes simplex virus-thymidine kinasegene (HSV-TK) is introduced using a retroviral expression vector.Methods to insert and express the HSV-TK gene and to activate acytotoxic prodrug such as ganciclovir are known (see e.g., U.S. Pat. No.6,576,464 issued to Gold and Lebkowski on Jun. 10, 2003 and U.S. Pat.No. 5,997,859 issued to Barber et al. on Dec. 7, 1999 which areincorporated herein by reference). To stop the growth of recombinant Bcells deemed unsafe or contributing to an adverse event the B cellsexpressing HSV-TK are provided with 20 μM ganciclovir (available asCytovene IV from Roche Laboratories, Nutley, N.J.). Conversion ofganciclovir into a toxic metabolite by the B cells expressing HSV-TKresults in their death. Cells not expressing HSV-TK are not harmed byganciclovir.

In another embodiment, chemical inducers of dimerization (CID) can beused, in which a proapototic molecule is adapted to encompass one ormore binding sites for a CID, which once reaching its target(s) causestheir oligomerization with ensuing activation of the apoptotic pathway.In this manner, different apoptotic pathways can operate as suicidesystems, including the death receptor Fas and the enzyme Caspase 9.Beside a very low risk for immunogenicity, these suicide genes share theadvantages of non cell-cycle dependency, full clinical compatibility andoptimal biodistribution, as CID are small molecule exquisitely designedfor suicide purposes. See, for example, J Cancer. 2011; 2: 378-382,which is incorporated herein by reference.

The isolated cell line can include an isolated B lymphocyte cell line oran isolated recombinant B lymphocyte cell line that recognizes one ormore antigens to an infectious bacterial or viral disease, e.g.,influenza antigen. Table 1 includes examples of protocols forconstructing the isolated B lymphocyte cell line or the isolatedrecombinant B lymphocyte cell lines including an exogenously-derivedand/or endogenously-derived membrane immunoglobulin andexogenously-derived and/or endogenously-derived secreted immunoglobulin.The secreted immunoglobulin from the isolated recombinant B lymphocytecell line can include one or more secreted anti-influenza broadlyneutralizing antibodies (Flu BNAb). The anti-influenza broadlyneutralizing antibodies can be directed to two or more epitopes on thesame influenza antigen (Flu BNAb1 and Flu BNAb2). The secretedanti-influenza immunoglobulin from the isolated recombinant B lymphocytecell line can include one or more secreted polyclonal antibodies (FluAbe) to the influenza antigen.

B Membrane Secreted Membrane Secreted lymphocyte ImmunoglobulinImmunoglobulin Immunoglobulin Immunoglobulin cell line 1 1 2 2 1 DNP-endog Flu exog Flu exog DNP- endog KLH BNAb BNAb KLH 2 DNP- exog Fluexog Flu exog Flu exog KLH BNAb1 BNAb1 BNAb2 3 DNP- exog Flu Ab_(n)endog Flu Ab_(n) endog None none KLH 4 Flu endog Flu Ab_(n) endog Fluexog Flu exog Ab_(n) BNAb BNAb

B lymphocyte protocol 1 is a protocol to produce isolated recombinant Blymphocytes. The protocol 1 immunizes a vertebrate subject with DNP-KLH(dinitrophenyl-Keyhole Limpet Hemocyanin) and select memory Blymphocytes including membrane immunoglobulin recognizing DNP andsecreted immunoglobulin recognizing DNP. Anti-DNP B lymphocytes can betransfected with nucleic acid vector including immunoglobulin genesencoding membrane and secreted anti-influenza broadly neutralizingantibody (BNAb).

The isolated recombinant anti-influenza B lymphocytes can be transferredto a vertebrate subject to protect the vertebrate subject from influenzainfection. The long-lived anti-influenza B lymphocytes can be activatedat will by injecting DNP-KLH into the vertebrate subject when flusymptoms arise or when a pandemic hits.

B lymphocyte protocol 2 is a protocol to produce isolated recombinant Blymphocytes. The protocol 2 isolates memory B lymphocytes from avertebrate subject. The isolated memory B lymphocytes are transfectedwith a nucleic acid vector including immunoglobulin genes encodinganti-DNP membrane immunoglobulin only and not anti-DNP secretedimmunoglobulin. B lymphocytes with anti-DNP membrane immunoglobulin canbe selected and transfected with immunoglobulin genes encoding twoanti-influenza BNAbs to two different epitopes of the influenza antigen.The immunoglobulin genes encoding each BNAb can encode membrane andsecreted forms of the BNAbs.

The isolated recombinant anti-influenza B lymphocytes can be transferredto a vertebrate subject to protect the vertebrate subject from influenzainfection. The long-lived anti-influenza B lymphocytes can be activatedat will to produce two anti-influenza BNAbs by injecting DNP-KLH intothe vertebrate subject when flu symptoms arise or when a pandemic hits.The long-lived anti-influenza B lymphocytes can also be activated atwill to produce two anti-influenza BNAbs by injecting influenza antigeninto the vertebrate subject. Unlike Protocol 1, no secretedimmunoglobulin to DNP-KLH will be produced when the B lymphocytes areactivated by DNP-KLH or by influenza antigen.

B lymphocyte protocol 3 is a protocol to produce polyclonal isolatedrecombinant B lymphocytes. The protocol 3 immunizes a vertebrate subjectwith influenza vaccine, e.g., tripartite seasonal influenza vaccine.Memory B lymphocytes that express membrane immunoglobulin recognizingthe influenza vaccine antigens are selected in the vertebrate subject.The selected polyclonal anti-influenza memory B lymphocytes aretransfected with immunoglobulin genes encoding an anti-DNP membraneimmunoglobulin.

The polyclonal anti-influenza B lymphocytes can be transferred to avertebrate subject to protect the vertebrate subject from influenzainfection. The polyclonal, long-lived anti-flu B cells can be activateden mass by injecting DNP-KLH into the vertebrate subject when flusymptoms arise or when a pandemic hits. In addition individual Blymphocyte clones can be activated by their cognate influenza antigen.

B lymphocyte protocol 4 is a protocol to produce polyclonal isolatedrecombinant B lymphocytes. The protocol 4 immunizes a vertebrate subjectwith influenza vaccine, e.g., tripartite seasonal vaccine. Memory Blymphocytes that express membrane immunoglobulin recognizing theinfluenza vaccine antigens are selected in the vertebrate subject.Polyclonal anti-influenza B lymphocytes are transfected withimmunoglobulin genes encoding anti-influenza BNAb in both membrane formand secreted form.

The isolated recombinant polyclonal anti-influenza B lymphocytes can betransferred to a vertebrate subject to protect the vertebrate subjectfrom influenza infection. The polyclonal, long-lived anti-influenza Blymphocytes can be activated en mass by injecting a full spectrum ofinfluenza vaccine antigens into the vertebrate subject when flu symptomsarise or when a pandemic hits. Each B lymphocyte produces a BNAb and aclone-specific immunoglobulin reactive with influenza.

In some aspects, the isolated B lymphocyte cell line including at leastone exogenously incorporated membrane immunoglobulin activated by thefirst antigen is capable of controlling expression of the at least oneendogenous secreted immunoglobulin reactive to the second antigen. Theexogenously incorporated membrane immunoglobulin acts as a receptor to aspecified ligand, e.g., the first antigen. Binding of the first antigento the exogenously incorporated membrane immunoglobulin controls signaltransduction through the exogenously incorporated membraneimmunoglobulin to control expression from the at least one endogenoussecreted immunoglobulin reactive to the second antigen. Binding of thefirst antigen to the exogenously incorporated membrane immunoglobulincontrols signal transduction through the membrane immunoglobulin tocontrol activation of the B lymphocyte or differentiation of the Blymphocyte.

FIG. 1 is a schematic of a diagrammatic view of hypotheticalimmunoglobulin genes for memory B lymphocytes. Heavy (H) chain gene ison chromosome 14. Kappa (κ) L chain gene is on chromosome 2. Lambda (λ)L chain gene is on chromosome 22. A functional allele and anonfunctional allele are present on chromosomes 14 and 2. Both λ L chainalleles are depicted as nonfunctional. As shown in Example 3, theimmunoglobulin genes encoding the H chain and L chain for an anti-PSAmembrane antibody are cloned in targeting plasmid vectors to allowtargeted integration in the corresponding nonfunctional Ig loci onchromosomes 14 and 2 respectively.

FIGS. 2A, 2B, and 2C are a schematic of a diagrammatic view ofnonfunctional and functional immunoglobulin heavy chain genes onchromosomes 14. The genetic structure of maternal chromosome 14 germlineconfiguration is shown in FIG. 2A. Exons for variable regions (V_(H)), Dsegments (D), J segments (J_(H)), IgM constant region (CHO, secretedtailpiece (TP) and the μ membrane anchor (TM and Cyt) are shown in FIG.2B. The genetic structure of paternal chromosome 14 functionallyrearranged is shown with recombined V, D and J segments (V_(H) D₁J₂) asshown in FIG. 2C. The genetic structure of secreted and membrane μ-Hchains encoded and alternate polyadenylation sites are shown. Note thatIg gene structure is simplified with only one constant region (C_(H))exon shown. Also promoter and enhancer sequences are omitted.

As shown in Example 3, the anti-PCLA immunoglobulin H and L chain genesare integrated into the Ig loci of the mature B cell which arefunctionally rearranged on chromosomes 14 and 2 respectively. See FIG.2B for functionally rearranged H chain locus.

FIGS. 3A, 3B, and 3C are a schematic of a diagrammatic view ofreplacement of immunoglobulin genes with heavy chain genes engineered toexpress membrane IgG and secreted IgG. The genetic structure of secretedand membrane γ-H chain gene with alternate polyadenylation sites areshown in FIG. 3A. The genetic structure of maternal chromosome 14 withan engineered membrane γ-H chain gene is shown in FIG. 3B. The geneticstructure of paternal chromosome 14 with an engineered secreted γ-Hchain gene is shown in FIG. 3C. Note that Ig gene structure issimplified with only one constant region (C_(H)) exon shown. Alsopromoter and enhancer sequences are omitted.

As shown in Example 3, the anti-PCLA IgG H chain gene (i.e., γ-H chaingene) may be engineered to remove coding sequences for the membranespanning domain (TM) and the cytoplasmic amino acids (Cyt) to yield aγ-H chain gene encoding a secreted H chain only (FIG. 3C).

FIGS. 4A, 4B, 4C, 4D are a schematic of a diagrammatic view of protocolsto produce recombinant B lymphocytes with membrane immunoglobulin to afirst antigen and secreted immunoglobulin to a second antigen. FIG. 4Ashows isolated memory B lymphocytes with endogenous DNA encodinganti-DNP membrane immunoglobulin and exogenous DNA encoding anti-Flubroadly neutralizing antibody (BNAb) secreted immunoglobulin. FIG. 4Bshows isolated memory B lymphocytes with exogenous DNA encoding anti-DNPmembrane immunoglobulin and exogenous DNA encoding anti-Flu broadlyneutralizing antibody (BNAb) secreted immunoglobulin. FIG. 4C showsisolated memory B lymphocytes with endogenous DNA encoding anti-Flu Abssecreted immunoglobulin and exogenous DNA encoding anti-DNP membraneimmunoglobulin. FIG. 4D shows isolated memory B lymphocytes withexogenous DNA encoding an anti-Flu BNAb secreted immunoglobulin andexogenous anti-DNP membrane immunoglobulin polypeptide delivered withliposomes.

FIG. 5 is a schematic of a diagrammatic view of a method 500 forproducing at least one immunoglobulin in an isolated B lymphocyte cellline 501 that includes isolating 502 from a vertebrate subject immunizedwith at least one second antigen, a B lymphocyte cell expressing atleast one endogenous secreted immunoglobulin reactive to the at leastone second antigen; introducing 503 into the isolated B lymphocyte cellat least one exogenous membrane immunoglobulin reactive to at least onefirst antigen to produce a recombinant B lymphocyte cell line; expandingand selecting 504 the isolated B lymphocyte cell line expressing themembrane immunoglobulin reactive to the at least one first antigen andexpressing the at least one endogenous secreted immunoglobulin reactiveto the at least one second antigen.

FIG. 6 is a schematic of a diagrammatic view of a method 600 forproducing at least one immunoglobulin in an isolated B lymphocyte cellline 601 that includes introducing 602 into at least one isolated Blymphocyte cell at least one exogenous membrane immunoglobulin reactiveto at least one first antigen to produce at least one first isolated Blymphocyte cell line; expanding and selecting 603 the at least one firstisolated B lymphocyte cell line expressing the membrane immunoglobulinreactive to the at least one first antigen; introducing 604 into the atleast one first isolated B lymphocyte cell line at least one exogenousnucleic acid encoding one or more secreted immunoglobulins reactive toat least one second antigen to produce at least one isolated recombinantB lymphocyte cell line; and selecting 605 the at least one isolatedrecombinant B lymphocyte cell line expressing the one or more secretedimmunoglobulin reactive to the at least one second antigen.

FIG. 7 is a schematic of a diagrammatic view of a method 700 forproducing at least one immunoglobulin in a recombinant B lymphocyte cellline 701 that includes isolating 702 from a vertebrate subject immunizedwith at least one first antigen, a B lymphocyte cell expressing at leastone endogenous membrane immunoglobulin reactive to the at least onefirst antigen; introducing 703 into the isolated B lymphocyte cell atleast one exogenous nucleic acid encoding at least one of a secretedimmunoglobulin reactive to at least one second antigen and expand thecell to produce a recombinant B lymphocyte cell line; and assaying 704for presence of the at least one exogenous secreted immunoglobulinreactive to the at least one second antigen to select the recombinant Blymphocyte cell line.

FIG. 8A illustrates a recombinant B cell receptor protein 1000 with asingle-chain variable fragment 1005 joined to IgG heavy chain domainsthat include a hinge segment 1008, joined to CH3 domain 1010,transmembrane 1015, and cytoplasmic 1020 domains attached to an IL-21receptor cytoplasmic domain 1025.

FIG. 8B includes an illustration of a recombinant B cell receptorexpression vector 1055 that includes a CMV promoter 1028 joined to arecombinant B cell receptor 1030 attached to a poly-A site 1035 and aneomycin resistance gene 1040.

FIG. 8C is an illustration of an active gamma heavy chain locus onchromosome 14 with perforin gene inserted 1105, including an Ig variablegene promoter 1045 joined to perforin cDNA 1048 and downstream an Igalpha constant region 1049, with transmembrane domain 1051 andcytoplasmic domain 1052.

FIG. 8D includes an illustration of a Sendai virus expression vectorwith transcription factor genes inserted 1150 including a viral NP gene1065 joined to a viral P/V gene 1062, with three transcription factorsT-bet 1064, RunX3 1066, and Eomes 1068 with the viral L gene 1075 at the3-prime end.

FIG. 9A illustrates the integration of a single chain (SC) antibody geneat the active, rearranged kappa light chain locus on human chromosome 2.An adenovirus-associated viral (AAV) vector bearing the gene for ananti-prostate cancer lipid antigen (PCLA) SC antibody flanked byhomology arms (HA) that target integration to the kappa constant regiongene (C kappa). The results of CRISPR-mediated integration of theanti-PCLA SC Antibody gene in the Ckappa gene on chromosome 2 are shown.

FIG. 9B shows the targeted integration of a chemokine receptor gene(CXCR3) at the active, rearranged immunoglobulin (Ig) mu heavy-chainlocus. An AAV vector bearing the gene for CXCR3 flanked by HA thattarget integration to the first exon (C_(H)μ1) of the Ig mu constantregion gene. The edited Ig mu heavy chain gene on chromosome 14 isshown.

FIG. 10 illustrates a method 1000 of activating the modified B cell 1070engineered with a predetermined exogenous or endogenous membraneimmunoglobulin 1060 to selectively engage a first target antigen 1050and subsequently secrete a predetermined antibody 1080 to a secondtarget antigen 1220 and/or secrete a reassigned biological agent and/orcytotoxic effector molecule(s) (1310) resulting in the death of targetcell(s) 1210.

Referring to FIG. 10, the modified B cell includes engaging or allowingto engage 1100 an antigen (either engineered or naturally occurring)with the predetermined surface immunoglobulin 1060. In the next step1200, receptor engagement 1100 leads to secretion of the predetermined(exogenously or endogenously incorporated) antibody 1080 configured toengage with the second target antigen 1220 on the target cell 1210.Finally, the next step 1300 includes the optional secretion of one ormore cytotoxic molecules (which may be reassigned biological agents)1310 for additional target cell destruction.

FIG. 11 illustrates a lymph node 1111 with modified B cells 1117 thatrecognize particular target antigens 1118 that contribute to activationof the modified B cells. As can be seen in the illustration, the medulla1115 is rich in macrophages and plasma cells, while the cortex 1114contains mostly inactivated B and T cells, as well as dendritic cellsand macrophages. In an embodiment, the modified B cells are implantedinto the lymph node 1111 for example, in the cortical area 1114 to beactivated, or even if they are already activated. As illustrated in FIG.11, the afferent lymph vessel 1113 and efferent lymph vessel 1112 bringsimmune system cells into and out of the lymph node 1111. In FIG. 11, thegerminal centers 1116 include locations for mature B cells toproliferate, differentiate, and generate antibody. In an embodiment, themodified B cells can be implanted into one or more germinal centers 1116of the subject's lymph node 1111.

In an embodiment, a B cell stimulation device 1205 can be implanted, forexample in the cortical area or capsular space of the lymph node 1111.The B cell stimulation device 1205 can include an array of wells 1208which can include, for example, different antigens and/or differentconcentrations of antigens and/or different adjuvants and/or differentconcentrations of adjuvants. For example, in one particular well 1206, apredetermined antigen 1207 (optionally with adjuvant) is remotelytriggered to be released in the lymph node and eventually thepredetermined antigen 1118 reaches the modified B cells 1117 forengagement. Such engagement can include priming (or being a “booster”)of the modified B cells, or can be an initial activation of the modifiedB cells, or if desired, can be a tolerizing of the modified B cells (forexample, with “self-antigen” related to an autoimmune disease). Asillustrated in FIG. 11, the B cell stimulation device 1205 can includeremotely triggering different antigens and/or concentrations over time1119 and can act on naturally occurring B cells as well as modified Bcells described herein. For example, over time 1119 the B cellstimulation device 1205 can release three different concentrationsand/or antigens into the blood stream or lymph node.

FIG. 12A illustrates insertion of a gene for interleukin 10 (IL-10) atthe first exon of the active, rearranged Ig heavy chain gene onchromosome 14. A rearranged Ig mu heavy chain gene with a variableregion (V_(H)DJ) exon, an intron and mu constant region (C_(H)μ) exonsis shown. An AAV vector encoding an interleukin 10 (IL-10) gene is shownwith a splice acceptor site (SA), a poly A addition site (pA) andflanking homology arms (HA) to target integration at the mu heavy chaingene. The edited Ig mu heavy chain gene is shown with the IL-10 genepositioned downstream of the variable region and interrupting the muconstant region gene.

FIG. 12B shows a lentiviral expression vector encoding a single chainmembrane antibody specific for an autoantigen, myelin oligodendrocyteglycoprotein (MOG). The vector contains a cytomegalovirus promoterelement (CMV) directing transcription of the single chain antibody. Thesingle chain antibody includes a single chain Fv (SCFV) segment fused toan Ig gamma constant region gene.

FIG. 13 illustrates integration of a bicistronic construct downstream(3′) of the V_(H) promoter and mu enhancer (μEnh) leading to disruptionof the active rearranged Ig gamma heavy chain gene on chromosome 14. Thelocation of guide RNAs (gRNA) to target CRISPR-mediated integration inthe intron and γCH1 exon of the gamma heavy chain gene are indicated.The class switch recombination site (CSRS) is indicated. An AAV vectorencoding a bicistronic construct is illustrated. The bicistronicconstruct includes genes for a recombinant B cell receptor (recBCR) andinterleukin 21 (IL-21) with self-cleaving peptides (P2a) encoded beforeeach gene. The edited gamma heavy chain gene is shown with thebicistronic construct expressed under the control of the V_(H) promoterand mu enhancer. Expression of the gamma H-chain gene is disrupted bythe bicistronic construct.

In a method for treating a disease in a vertebrate subject with animmunotherapeutic product, the recombinant B lymphocyte cell line may beautologous to one of the one or more vertebrate subjects. Alternatively,in a method for treating a disease in a vertebrate subject with animmunotherapeutic product the recombinant B lymphocyte cell line may beallogeneic to one of the one or more vertebrate subjects. In the casewhere the recombinant B lymphocyte cell line is allogeneic to one of theone or more vertebrate subjects. In each case when necessary, therecombinant B lymphocyte cell line can be modified to reduce oreliminate expression of MHC Class I (MHC I) proteins or mismatched HLAantigens in the recombinant B lymphocyte cell line to avoid allograftrejection and to reduce or eliminate a graft versus host disease in therecipient of the allogeneic recombinant B lymphocyte cells. See, e.g.,U.S. application Ser. No. 12/804,650, and U.S. application Ser. No.12/804,647, which are incorporated herein by reference.

A vertebrate subject is treated with unmatched, allogeneic donorrecombinant B lymphocyte cells engineered to block the presentation ofMajor Histocompatibility Class I (MHC I) proteins on their cell surface.Allogeneic donor recombinant B lymphocyte cells are transfected with alentiviral expression vector that directs the expression of a microRNA(miRNA) that inhibits beta2-microglobulin ((32M) protein translation andblocks MHC I assembly and presentation on the cell surface. Thegenetically engineered recombinant B lymphocyte cells are injected intothe patient. The inhibition of MHC I production in engrafted recombinantB lymphocyte cells is controlled by a regulatory module and an effectormolecule, doxycycline. In the event that the recombinant B lymphocytecells must be eradicated, doxycycline is administered to repressexpression of the miRNA, thereby allowing expression of β₂M and MHC I onthe cell surface and evoking an alloreactive immune response.

A vertebrate subject is treated with recombinant B lymphocyte cells thathave reduced expression of Major Histocompatibility Class I (MHC I)proteins on their cell surface, in order to avoid immune rejection ofthe transplanted cells. The engineered recombinant B lymphocyte cellsalso contain a suicide mechanism that can be activated by theadministration of a prodrug, ganciclovir, in the event of uncontrolledproliferation or other adverse events associated with the recombinant Blymphocyte cells.

A vertebrate subject is treated with a recombinant B lymphocyte cellsthat are modified to reduce their expression of mismatched HLA antigensand thus avoid allograft rejection. Recombinant B lymphocyte cells areinfected with a lentivirus vector encoding microRNA (miRNA) thatinhibits the expression of specific donor HLA alleles not shared by therecipient. Production of mismatched HLA-A, -B, -C, -DRBI, and -DQB1alleles are blocked by miRNAs, and the corresponding HLA proteins arenot expressed by the modified donor recombinant B lymphocyte cells.

A vertebrate subject is treated by transplantation with recombinant Blymphocyte cells. Allogeneic recombinant B lymphocyte cells are modifiedto reduce expression of MHC Class I (MHC I) proteins by expression of aviral gene that targets MHC I proteins for destruction. Recombinant Blymphocyte cells are transduced with a lentiviral expression vectorencoding cytomegalovirus (CMV) protein, unique sequence 11 (US11), totarget MEW I proteins for destruction and avoid allograft rejection (seee.g., Lin et al., Cellular and Molecular Immunology 4: 91-98, (2007),which is incorporated herein by reference).

In addition to secreting antibodies, as described herein, in anembodiment the one or more modified B lymphocyte cells have beenengineered to secrete a non-antibody protein (e.g., glycoprotein,proteoglycan, amino acid, etc.) when prompted by engagement with thesurface immunoglobulin with a specifically designed “trigger” antigen.For example, the non-antibody protein may include a neurotransmitter,hormone, cytokine, fat, vitamin, mineral, or anti-inflammatory agent.

In an embodiment, one or more neurotransmitters, such as dopamine,serotonin, acetylcholine, GABA, norepinephrine, oxytocin, etc., aresecreted by the modified B lymphocyte cells.

In an embodiment, one or more neurotransmitters are secreted by one ormore modified B lymphocyte cells upon ingestion, injection,implantation, or otherwise a transfer of the modified B lymphocytes to asubject. For example, it has been documented that naturally occurring Blymphocytes will home to Peyer's patches and other areas of thegastrointestinal tract upon oral ingestion (e.g., by way of breastmilk).See, for example, Cabinian et al, PLoS One. 2016; 11(6): e0156762, whichis incorporated herein by reference.

Furthermore, it is known that the microbiome plays a role in cancerdevelopment, progression, and therapy. See, for example, Bhatt et al, CACancer J Clin 2017; 67:326-344, which is incorporated herein byreference.

Thus, in an embodiment, an oral or other formulation of a compositionincluding one or more modified B lymphocytes as described herein can beprovided to a subject for treatment of a disease or indication. In anembodiment, a composition can be delivered in another route, for exampleas an intramuscular injection, subcutaneous injection, sublingualadministration, buccal administration, parenteral administration, analadministration, intralymphatic administration, or another route ofadministration that is sufficient to provide delivery of the compositionto the subject for treatment.

In an embodiment, an oral or other formulations of composition includingone or more modified B lymphocytes further includes one or more life,dead, or preserved strains of microorganism, such as E. coli,Bacteroides, Bifidobacterium, Bacillus, Saccharomyces, Prevotellatanneraie, Neisseria lactamica, Streptococcus, Staphylococcus, Serratia,Corynebacteria, Lactobacillus, or others. For example, in an embodiment,strains such as L. acidophilus, B. longum, B. bifidum, B. lactis, B.infantis, B. animalis, L. rhamnosus, L. fermentum, L. plantarum, L.brevis, L. salivarius, L. paracasei, L. gasseri, L. reuteri, B.coagulans, S. salivarius, etc.

In an embodiment, the modified B cells provide surveillance in thesubject's body, such that the cells are directed to secrete a specificantigen when the cell's surface immunoglobulin receptor is triggeredwith the antigen selected for in the process of engineering the cells.

In order to selectively stimulate and re-stimulate the modified B cells,a particular antigen (e.g., DNP-KLH) is administered in the absence ofadjuvant. The endogenous response of these modified B cells should belimited to stimulation without over-stimulation that could result intolerance to the antigen rather than antibody secretion reaction.Further, a DNP conjugate of a peptide from KLH would likely elicit arestricted endogenous response.

In an embodiment, the modified B cells are stimulated or restimulated touse an alternate carrier (e.g., DNP-Human Serum Albumin) which shouldnot elicit an immune response but will provide a “booster” antigenstimulation to the modified B cells.

In an embodiment, a B cell stimulation device is implanted in or on thesubject in order to provide stimulation or re-stimulation of themodified B cells (or of a patient's own innate B cells). For example,the B cell stimulation device can include a microarray or microchipdevice with antigen (and optionally adjuvant, cytokines, chemokines,etc.) on or in the device and can be injected into a subject such thatactivating the antigen-carrying device allows for release of the antigenand/or adjuvant for “boosting” the activation of the modified B cells.

For example, a microchip containing one or more small, hermeticallysealed compartments can be activated by remote control (eg wirelesssignal) to trigger the release of one or more compartments. In anembodiment, the one or more compartments can be triggered to release theantigen and/or adjuvant and/or other molecules (eg cytokines,chemokines, growth factors, etc.) based on a pre-programmed dosingschedule. In an embodiment, the one or more compartments can betriggered as desired. In an embodiment, the subject can control thetrigger for the B cell stimulation device. In an embodiment anotherentity can control the trigger for the subject's B cell stimulationdevice. In an embodiment, the B cell stimulation device is placed inlymphatic tissue of a subject, including but not limited to, forexample, lymph nodes, GALT, MALT, spleen, liver, etc. See FIG. 11.

In an embodiment, the B cell stimulation device can be easily implantedand/or removed in a healthcare setting. In an embodiment, the B cellstimulation device can include at least ten, at least twenty, at leastfifty, at least one hundred, at least two hundred, or more doses forstimulating B cells. In an embodiment, each dose is the same in aparticular B cell stimulation device. In an embodiment, multipledifferent doses include different contents (eg different antigen and/ordifferent adjuvant). In an embodiment, the compartments of the B cellstimulation device is configured such that each dose can be released ata specific time (eg by predetermined program, by way of sensorsdetecting a specific physiological parameter that causes or warrantsrelease of a dose, or by active intervention by the subject or anotherentity such as a healthcare worker or computing device).

In an embodiment, the B cell stimulation device includes multipledifferent antigens and/or multiple different adjuvants and/or cytokinesor other molecules such as ligands or transcription factors (configuredas each in its own compartment or a mix of two or more in a singlecompartment) and can be wirelessly released into the subject's body. Inan embodiment, each compartment is independently addressable andindependently activatable in any desired sequence of release. In anembodiment, the B cell stimulation device includes electronic circuitryincluding wireless communications (eg radio frequency), circuitry inelectronic communication with each compartment for independent releaseof its contents, timer or clock for accurate interval spacing and/orrelease of contents of a compartment, and a controller in electroniccommunication with the various electrical components for properfunctioning.

For example, a wirelessly controlled implantable microchip-based drugdelivery device for delivering human parathyroid hormone fragment hasbeen successfully tested in clinical trials in humans as a bioequivalentto daily injections. See, for example, Farra, Science Translational Med22 Feb. 2012, Vol. 4, Issue 122, pp. 122ra21, which is incorporatedherein by reference.

In an embodiment, a B cell stimulation device similar to the microchipdevice described in the citation above is configured for stimulating Bcells in a subject, including the modified B cells described hereinthroughout. For example, a microchip array device with discretecompartments with an impermeable, thin metallic membrane is configuredto retain the contents in a lyophilized form or other activatable form.For example, the metallic membrane can be removed by electrothermalablation, which releases the contents of the compartment in a controlledmanner.

In an embodiment, a B cell stimulation device is inserted subcutaneouslyinto a subject that has already received or will receive modified Bcells as described herein. In an embodiment, the B cell stimulationdevice is implanted into a lymphoid tissue of a subject that has alreadyreceived or will receive modified B cells as described. Lymphoid tissuecan include, for example, lymph nodes, tonsils, spleen, Peyer's patches,mucosa associated lymphoid tissue, bone marrow, or thymus.

In an embodiment, a B cell stimulation device can be approximately 50mm×30 mm×10 mm (l×w×h), as described in Farra herein above, with twomicrochips with 10 reservoirs each. In an embodiment, the stimulationdevice can be approximately half this size, with a single microchip with10 reservoirs each. In an embodiment, the stimulation device can be muchsmaller, for example, a single microchip with 1, 2, 3, 4, 5, 6, 7, 8, or9 reservoirs.

In an embodiment, the B cell stimulation device includes at least onecompartment with an enzyme, such as collagenase, to assist withpenetration of the fibrous membrane capsule that can form around anyimplant in a subject's body. Typically, the fibrous tissue capsule isless than one mm thick and allows for passage of molecules through it.Id. However, if additional penetration is needed, enzymatic releaseprior to release of B cell stimulants can be performed. In this way, oneor more compartments containing collagenase or other enzymes arereleased prior to release of the B cell stimulants (eg antigen,adjuvant, cytokine, other immune stimulant, etc.).

In a report of a patient who underwent CAR-T cell immunotherapy, it wasnoted that the patient attained complete remission of brain metastases,and upon physical biopsy of a recurrent, subcutaneous lesion, the CAR-Tcells became activated or re-activated with the result that thesubcutaneous tumor also regressed. See Science News, Aug. 28, 2017,online report from Massachusetts General Hospital, which is incorporatedherein by reference. In an embodiment, the modified B cells describedherein are stimulated or re-stimulated by physical biopsy sampling ofsuspected tumor tissue, or lymph node.

PROPHETIC EXAMPLES Example 1

Recombinant Memory B Lymphocytes that Express Two DifferentAntibodies: 1) a B Cell Receptor (BCR) that Recognizes a Model Antigen,Dinitrophenol-Keyhole Limpet Hemocyanin (DNP-KLH), and 2) A SecretedAntibody that Neutralizes Multiple Strains of Influenza Virus.

An isolated recombinant B lymphocyte cell line that produces a secretedbroadly neutralizing immunoglobulin to influenza virus and produces amembrane immunoglobulin to a model antigen can be utilized for celltherapy in a mammalian subject. The recombinant B lymphocyte cell linecan be injected into the mammalian subject as cell therapy to provideimmunological protection from infection by influenza virus. Therecombinant B lymphocyte cell line can be activated in vivo or ex vivoto produce the broadly neutralizing influenza antibody by injecting themammalian subject (or an in vitro cell culture) with model antigen,dinitrophenol-keyhole limpet hemocyanin (DNP-KLM). The timing tostimulate immunological protection from influenza virus infection in themammalian subject can be chosen based upon the timing of an outbreak ofinfluenza infection in the population at large.

An individual is immunized with a model antigen, dinitrophenol (DNP), toelicit memory B cells with B cell receptors (BCR) specific for the DNPmodel antigen. Memory B cells develop in response to immunization withDNP conjugated to a carrier protein, keyhole limpet hemocyanin (KLH). Aprimary immunization with 1 mg of DNP-KLH (see e.g., BiosearchTechnologies DNP-KLH Product Info Sheet which is incorporated herein byreference) is injected subcutaneously in the right arm. See e.g.,Rentenaar et al., Kidney International 62: 319-328, 2002 which isincorporated herein by reference. Approximately 12-14 days afterimmunization memory B cells expressing BCR specific for DNP are isolatedusing dinitrophenol-human serum albumin-biotin (DNP-HSA-biotin) andphycoerythrin-streptavidin (available from Biosearch Technologies,Novato, Calif.) and a fluorescein-anti-CD27 antibody to identify memoryB cells. DNP-specific memory B cells are isolated by cell sorting with afluorescence activated cell sorter (e.g., FACSAriaIII® available fromBecton Dickinson, Franklin Lakes, N.J.). For example, see U.S. Pat. No.7,378,276 issued to Ettinger et al. on May 27, 2008 and U.S. Pat. No.7,993,864 issued to Brown et al. on Aug. 9, 2011 which are incorporatedherein by reference.

Memory B cells expressing BCRs that binds DNP are genetically engineeredto express a secreted antibody which is a broadly neutralizing antibodyreactive with multiple strains of influenza. Memory B cells expressinganti-DNP BCRs, containing membrane IgG antibodies, have a productivelyrearranged and expressed membrane immunoglobulin heavy (H) chain genewhich resides on chromosome 14 (one of two parental chromosome 14copies). However, the other parental chromosome 14 has an immunoglobulin(Ig) H chain gene that is not productively rearranged and not expressed.See FIGS. 1, 2A and 2B. This phenomenon, termed “allelic exclusion”,yields individual B cells which express only one Ig heavy chain (and oneIg light (L) chain) and thus only one antibody (see e.g., Abbas et al.,Cellular and Molecular Immunology, 7^(th) Ed., Elsevier Saunders,Philadelphia, Pa., 2012 which is incorporated herein by reference). Tocreate B cells producing two different antibodies the memory B cellsexpressing anti-DNP BCRs are modified by replacing the non-functional,non-expressed immunoglobulin genes with functional, expressedimmunoglobulin genes (for H and L chain). For example, the replacementimmunoglobulin genes may encode a secreted antibody, which is a broadlyneutralizing anti-influenza antibody.

In an embodiment, the IgH chain or IgL chain chromosomal loci arenon-expressed Ig alleles where, for example, the endogenous rearrangedVH promoter proximal to the mu enhancer is utilized for expression (dueto V-J joining, the non-expressed allele will likely not have a VHpromoter proximal to the Constant region).

The immunoglobulin genes encoding a broadly neutralizing antibodyreactive with multiple strains of influenza virus may be isolated fromthe chromosomal DNA of a human B cell clone that produces the antibody.For example human B cells isolated from an individual who is immune toinfluenza virus infection are immortalized by infecting the isolated Bcells with Epstein Barr virus (EBV). Supernatants derived fromindividual EBV-transformed B cell clones are tested in an immunoassayfor antibodies that recognize influenza virus. Methods to immortalize Bcells and to detect anti-viral antibodies are described (see e.g., Zhanget al., Proc. Natl. Acad. Sci. USA 107: 732-737, 2010 and Corti et al.,J. Clin. Investigation 120: 1663-1673, 2010 which are incorporatedherein by reference).

Methods to clone Ig heavy (H) chain and light (L) chain genes may beused. See e.g., U.S. Pat. No. 7,741,077 issued to Grawunder et al. onJun. 22, 2010 and Early et al., Proc. Natl. Acad. Sci. USA 76: 857-861,1979 which are incorporated herein by reference. For example, anEBV-transformed B cell line expressing a human anti-influenza antibody,IgG₁(kappa), is grown in culture and used as a source to isolatemessenger RNA (mRNA) and genomic DNA using standard methods employingphenol/chloroform. See e.g., Sambrook et al., In: Molecular Cloning: ALaboratory Manual, 2^(nd) Ed., Cold Spring Harbor Press, Cold SpringHarbor, N.Y., 1989. The mRNA encoding the IgG₁ H chain and the kappa Lchain are molecularly cloned following amplification using thepolymerase chain reaction (PCR) and reverse transcriptase (RT). Methodsand Ig gene primers to amplify Ig H chain mRNA and Ig L chain mRNA aredescribed in U.S. Pat. No. 7,741,077 Ibid. The H and L chain mRNA(amplified as complementary DNA) are cloned in a plasmid vector (e.g.,pCR®2.1-TOPO plasmid available from Invitrogen Corp., Carlsbad, Calif.).The DNA sequence of the Ig H chain variable (V) region (including theVh, D and J segments) and the kappa L chain V-region (including the Vkand Jk segments) are determined. The V-region DNA sequences may bedetermined by automated DNA sequencing (DNA sequencing services areavailable from Charles River Laboratories International, Inc.,Wilmington, Mass.).

To isolate the corresponding genomic Ig genes, the genomic DNA isolatedfrom the anti-influenza B cell line (see above) is used as a templatefor PCR amplification of the human H chain gene and kappa L chain gene.PCR primers (oligonucleotides) to amplify the V-region genes, (includingtheir respective promoters and flanking regions upstream (i.e., 5′ ofthe V genes) are determined by searching a human genome database withthe V-region DNA sequences established from the cloned Ig mRNA. Forexample, a human genome nucleotide database available from the NationalCenter for Biotechnology Information can be searched with a computerprogram, BLAST, for sequences matching the H- and L-chain V-regions. AHuman RefSeq Genome database and BLAST software are available online(see e.g., the world wide web at blast.ncbi.nlm.nih.gov/Blast.cgi).Primers to amplify the Ig constant regions, enhancer sequences, theH-chain membrane anchors, poly A addition sites and downstream flankingregions (i.e., 3′ of the Ig genes) are described (see e.g., U.S. Pat.No. 7,741,077 Ibid.). The PCR-amplified, genomic fragments can be clonedin a plasmid vector such as pCR®2.1-TOPO available from InvitrogenCorp., Carlsbad, Calif.).

Memory B cells expressing anti-DNP membrane IgG are engineered toexpress Ig genes encoding a secreted IgG antibody specific forinfluenza. The anti-influenza IgG₁ H chain gene (i.e., γ₁-H chain gene)may be engineered to remove coding sequences for the membrane spanningdomain (TM), the cytoplasmic amino acids (Cyt), and a polyA additionsite to yield a γ₁-H chain gene encoding a secreted H chain only. SeeFIG. 3C and Abbas et al., Ibid. Ig genes are engineered using standardmethods in molecular biology (see e.g., Sambrook et al., In: MolecularCloning: A Laboratory Manual, 2^(nd) Ed., Cold Spring Harbor Press, ColdSpring Harbor, N.Y., 1989 which is incorporated herein by reference) toremove the membrane exons and to retain the promoter and enhancersequences associated with the functional anti-influenza Ig genes (seee.g., Abbas et al., Ibid.). The Ig H chain and Ig L chain genes encodingthe antiviral antibody may be inserted in the non-expressed Ig gene lociby using methods of homologous recombination (see e.g., U.S. Pat. No.5,202,238 issued to Perry et al. on Apr. 13, 1993; U.S. Pat. No.6,570,061 issued to Rajewsky and Zou on May 27, 2003 and U.S. Pat. No.6,841,383 issued to Reff et al., on Jan. 11, 2005 which are incorporatedherein by reference). Methods to identify and target DNA sequences ofindividual Ig gene loci in the memory B cells are known (see e.g., Suket al., Genome Research published online Aug. 3, 2011.DOI/10.1101/gr.125047.111 which is incorporated herein by reference).DNA sequences determined from the nonexpressed immunoglobulin loci(i.e., nonfunctional immunoglobulin genes) are used to targetrecombination with the anti-influenza immunoglobulin genes.

To promote homologous recombination the Ig genes encoding the H chainand L chain for a secreted anti-influenza antibody are cloned in plasmidtargeting vectors to obtain targeted integration in the correspondingnonfunctional, germline Ig loci on chromosomes 14 and 2 respectively.See FIG. 1 and FIG. 2A. For example, DNA sequences 5′ of the J_(H)segments (see FIG. 2A) are cloned upstream (5′) of the anti-influenzaγ₁-H chain gene in a targeting plasmid, and sequences downstream (3′) ofthe μ-H chain membrane anchor exons are cloned downstream (3′) of theγ₁-H chain gene to promote recombination at the germline H-chain locuson chromosome 14. Methods for construction of targeting vectorscontaining target sequences, replacement genes and selectable markersare described (see e.g., U.S. Pat. No. 5,202,238 Ibid., U.S. Pat. No.6,570,061 Ibid., and U.S. Pat. No. 6,841,383 Ibid.).

Targeting vectors encoding a secreted anti-influenza antibody are usedto replace the nonfunctional, germline μ-H chain gene and thenonfunctional kappa L chain gene in memory B cells expressing membraneanti-DNP IgG. The targeting vector plasmids are linearized byrestriction enzyme digestion and transferred by electroporation into thememory B cells followed by selection for the targeting vector plasmids.Methods and reagents for electroporation of primary mammalian cells aredescribed (see e.g., “Electroporation Guide” available from BioRad Inc.,Hercules, Calif. which is incorporated herein by reference). Memory Bcells, following electroporation, are cultured in tissue culture mediacontaining selection drugs, such as G418 and methotrexate, to select forselectable marker genes, i.e., neomycin resistance gene anddihydrofolate reductase, respectively, present on the H and L chaintargeting vectors. Selectable marker genes and their use are described(see e.g., U.S. Pat. No. 6,841,383 Ibid.). Electroporated memory B cellswith resistance to both G418 and methotrexate are tested for expressionof secreted IgG which binds influenza. Following transfection andselection of the memory B cells, those cells producing secreted IgGantibodies specific for influenza are identified using standardimmunoassays to assess B cell supernatants (see e.g., Zhang et al.,Proc. Natl. Acad. Sci. USA 107: 732-737, 2010 which is incorporatedherein by reference).

To insure the recombinant memory B cells are safe for use in patients asuicide gene is introduced into the B cells. To stop uncontrolledproliferation (and other adverse events) by the recombinant memory Bcells, a suicide gene, Herpes simplex virus-thymidine kinase gene(HSV-TK) is introduced using a retroviral expression vector. Methods toinsert and express the HSV-TK gene and to activate a cytotoxic prodrugsuch as ganciclovir are known (see e.g., U.S. Pat. No. 6,576,464 issuedto Gold and Lebkowski on Jun. 10, 2003 and U.S. Pat. No. 5,997,859issued to Barber et al. on Dec. 7, 1999 which are incorporated herein byreference). To stop the growth of recombinant B cells deemed unsafe orcontributing to an adverse event the B cells expressing HSV-TK areprovided with 20 μM ganciclovir (available as Cytovene IV from RocheLaboratories, Nutley, N.J.). Conversion of ganciclovir into a toxicmetabolite by the B cells expressing HSV-TK results in their death.Cells not expressing HSV-TK are not harmed by ganciclovir.

The recombinant memory B cells may be activated and expanded in vitro toassess their proliferation, activation and production of the secretedanti-influenza antibody. Engineered anti-DNP memory B cells isolated asdescribed above are cultured with DNP-HSA in vitro to activate thecells. For example, memory B cells at about 10⁵ to10⁶ cells/mL arecultured in tissue culture flasks in standard media (e.g., RPMI 1640serum-free media available from Sigma-Aldrich Chem. Co., St. Louis, Mo.)which contain approximately 1 μg/ml of DNP-HSA. Methods to activatememory B cells are described (see e.g., U.S. Pat. No. 7,378,276 Ibid.).To assess activation the cells are tested in a proliferation assay after3-5 days in culture. Aliquots of the culture are supplemented with³H-thymidine and cultured an additional 16 hours. ³H-thymidine uptake ismeasured by using a liquid scintillation counter (see e.g., U.S. Pat.No. 7,378,276 Ibid.). Equivalent cultures of memory B cells withoutDNP-HSA serve as negative controls for the proliferation assay. Toassess antibody production by the activated memory B cells culturesupernatants derived from 3-5 day cultures are tested by enzyme-linkedimmunosorbent assay (ELISA) to detect and quantitate the anti-influenzaantibody. Methods to detect and quantitate anti-influenza antibodieswith ELISA are described (see e.g., Khurana et al., PLoS Med. publishedonline Apr. 21, 2009; doi:10.1371/journal.pmed.1000049 and Corti et al.,Science 333: 850-856, 2011 which are incorporated herein by reference).Purified anti-influenza antibody derived from recombinant cell lines(see e.g., Wrammert et al., Nature 453: 667-671, 2008 which isincorporated herein by reference) may be used to create standard curvescorrelating absorbance and antibody concentration in ELISA assays.Supernatants from non-activated, recombinant memory B cells (i.e.cultured without DNP-HA) serve as negative control samples for theanti-influenza antibody ELISA.

The recombinant B lymphocyte cell line can be activated in vivo or exvivo to produce the secreted broadly neutralizing influenza antibody byinjecting the mammalian subject (or an in vitro cell culture) with modelantigen, DNP-KLH, to activate production of the secreted antibody fromthe recombinant B lymphocyte cell line. Timing to stimulateimmunological protection from influenza virus infection in the mammaliansubject can be chosen based upon the timing of an outbreak of influenzainfection in the population at large.

Example 2 Memory B Lymphocytes Engineered to Express a B Cell Receptor(BCR) Recognizing Dinitrophenol (DNP) and a Secreted AntibodyRecognizing Hepatitis C Virus

An isolated recombinant B lymphocyte cell line that produces a secretedimmunoglobulin against hepatitis C virus (HCV) and produces a membraneimmunoglobulin to a model antigen can be utilized for cell therapy in amammalian subject. The recombinant B lymphocyte cell line can beinjected into the mammalian subject as adoptive cell therapy to provideimmunological protection from infection by hepatitis C virus. Therecombinant B lymphocyte cell line can be activated in vivo or ex vivoto produce secreted anti-HCV antibody by injecting the mammalian subject(or an in vitro cell culture) with model antigen, dinitrophenol-keyholelimpet hemocyanin (DNP-KLH). Timing to stimulate immunologicalprotection from HCV infection in the mammalian subject can be chosenbased upon the timing of exposure of the mammalian subject to HCV orbased upon the appearance of symptoms in the subject.

Memory B cells expressing membrane IgG, also known as surface IgG or Bcell receptor (BCR), are isolated from the peripheral blood of a patientwith a chronic hepatitis C virus (HCV) infection. Polyclonal memory Bcells are isolated from the patient's peripheral blood: 1) by isolatingperipheral blood mononuclear cells using Ficoll Hypaque densitygradients (available from Sigma Aldrich, St. Louis, Mo.); 2) by negativeselection of total B cells using magnetic beads (available from StemCell Technology, Vancouver, BC), and 3) by labeling the cells withfluorescent monoclonal antibodies that recognize membrane IgG and CD27,a memory B cell marker, and performing fluorescence-activated cellsorting. See for example, U.S. Pat. No. 7,378,276 issued to Ettinger etal. on May 27, 2008 and U.S. Pat. No. 7,993,864 issued to Brown et al.on Aug. 9, 2011 which are incorporated herein by reference.

Immunoglobulin (Ig) genes encoding a membrane IgG antibody specific forDNP may be obtained from healthy volunteers who are immunized withDNP-KLH (see e.g., Biosearch Technologies: DNP-KLH Product Info Sheetwhich is incorporated herein by reference). Memory B cells with membraneIgG recognizing DNP are isolated by cell sorting with afluorescence-activated cell sorter (e.g., FACSAriaIII® available fromBecton Dickinson, Franklin Lakes, N.J.). For example, see U.S. Pat. No.7,378,276 Ibid. and U.S. Pat. No. 7,993,864 Ibid. Ig genes encoding ananti-DNP antibody are isolated from individual B cells (see e.g., Tilleret al., J. Immunol. Methods 329: 112-124, 2008 which is incorporatedherein by reference). For individual anti-DNP B cells the Ig heavy (H)and corresponding Ig light (L) chain gene transcripts are amplified byreverse transcriptase-polymerase chain reaction (RT-PCR) usingSuperscript®III reverse transcriptase (available from Invitrogen Corp.,Carlsbad, Calif.) and Taq DNA polymerase (available from Qiagen,Valencia, Calif.). Reaction conditions and oligonucleotide primers toamplify Ig H chains and Ig L chains are known (see e.g. Tiller et al.,Ibid.). The DNA fragments encoding the Ig H and L chain variable (V)region genes are isolated and cloned in mammalian expression vectorscontaining Ig H and L chain constant region genes (e.g., Cγ₁ and Ck).DNA sequences of the cloned anti-DNP Ig genes (γ₁-H chain and κ-L chain)are determined using a DNA sequencer (e.g., using a 3130 GeneticAnalyzer available from Applied Biosystems, Carlsbad, Calif.). The IgG₁H chain gene (i.e., γ₁-H chain gene) is engineered to remove the “tailpiece” and polyadenylation site encoding the secreted form of the Hchain, thus only a membrane γ₁-H chain is encoded by the engineered gene(see e.g., FIG. 3B, and Abbas et al., Cellular and Molecular Immunology,7^(th) Ed., Elsevier Saunders, Philadelphia, Pa., 2012 which isincorporated herein by reference). For example, the cloned γ₁-H chaingene may be amplified by PCR with primers that amplify the γ₁-H chainconstant region gene but omit the tail piece and polyadenlyation siteencoding the secreted form of the γ₁-H chain (see FIG. 3B). The primermay also add a RNA splice donor site to the 3′ end of the γ₁-H chaingene and a unique restriction enzyme site (e.g., a site for Not I;enzyme available from New England Biolabs, Ipswich, Mass.). A separateDNA fragment encoding a RNA splice acceptor site, the membrane anchorexons, and the remainder of the γ₁-H chain gene are PCR-amplified usingPCR primers containing restriction enzyme sites which allow reassemblyof the γ₁-H gene encoding a membrane form of the γ₁-H chain. See FIG.3B. Methods to amplify and assemble Ig genes are described (see e.g.,U.S. Pat. No. 7,741,077 Ibid.).

Memory B cells obtained from a patient with a chronic HCV infection aregenetically engineered by replacing their functional, expressed Ig geneswith Ig genes encoding a membrane IgG(κ) which recognizes DNP (seeabove). The Ig H and L chain genes encoding the anti-DNP antibody may beinserted in the functional, expressed Ig gene loci on chromosomes 14 and2 by using methods of homologous recombination (see e.g., U.S. Pat. No.5,202,238 issued to Perry et al. on Apr. 13, 1993; U.S. Pat. No.6,570,061 issued to Rajewsky and Zou on May 27, 2003 and U.S. Pat. No.6,841,383 issued to Reff et al., on Jan. 11, 2005 which are incorporatedherein by reference). To target integration into the functional γ1-Hchain locus, targeting sequences from the intron between the J₁ clusterand the μ constant region gene (C_(H)μ; see FIG. 2A) are placed 5′ ofthe anti-DNP γ-H chain gene and sequences downstream from the γ1membrane anchor exons are placed 3′ of the γ-H chain gene (see FIG. 3A).Analogous targeting sequences (i.e., from the Jk-Ck intron and 3′ of theCk gene) are used for targeting the anti-DNP kappa L chain gene into thefunctional Ck gene. The targeting vectors for anti-DNP H and L chaininclude selectable marker genes, e.g., hygromycin resistance andhistidinol dehydrogenase, respectively. Media containing hygromycin andhistidinol are used to select for engineered mature B cells expressingsecreted IgG anti-DNP antibody. Essential transcriptional promotersequences and enhancer sequences necessary for Ig gene expression areretained in the Ig gene integrants (see Abbas et al., Ibid.). Followingtransfection and selection of the memory B cells, those cells producingmembrane IgG antibodies specific for DNP are isolated using DNP-KLHattached to magnetic beads (protocols and separation devices areavailable from Miltenyi Biotec, Auburn, Calif.).

To create B cells producing two different antibodies the engineeredmemory B cells expressing an anti-DNP membrane IgG are engineered toreplace their non-functional, germline Ig genes with functional Ig genes(for H and L chain). For example, the replacement Ig genes may encode asecreted antibody, an anti-HCV antibody. The Ig genes encoding ananti-viral HCV antibody may be isolated from the chromosomal DNA of ahuman B cell clone that produces the antiviral antibody. For example,human B cells from an individual immune to HCV are immortalized byinfection with Epstein Barr virus (EBV) and supernatants derived fromindividual B cell clones are tested in an immunoassay for antibodiesthat recognize HCV. Methods to immortalize B cells and to detectanti-viral antibodies are described (see e.g., Zhang et al., Ibid. andCorti et al., J. Clin. Investigation 120: 1663-1673, 2010 which isincorporated herein by reference).

Methods may be used to clone Ig heavy (H) chain and light (L) chaingenes. (see e.g., U.S. Pat. No. 7,741,077 issued to Grawunder et al. onJun. 22, 2010 and Early et al., Proc. Natl. Acad. Sci. USA 76: 857-861,1979 which are incorporated herein by reference). For example, anEBV-transformed B cell line expressing a human anti-HCV antibody,IgG₁(kappa), is grown in culture and used as a source to isolatemessenger RNA (mRNA) and genomic DNA using standard methods employingphenol/chloroform (see e.g., Sambrook et al., In: Molecular Cloning: ALaboratory Manual, 2^(nd) Ed., Cold Spring Harbor Press, Cold SpringHarbor, N.Y., 1989). The mRNA encoding the IgG₁ H-chain and the kappaL-chain are molecularly cloned following amplification using thepolymerase chain reaction (PCR) and reverse transcriptase (RT). Methodsand Ig gene primers to amplify the H chain and L chain mRNA aredescribed in U.S. Pat. No. 7,741,077 Ibid. The H and L chain mRNA(amplified as complementary DNA) are cloned in a plasmid vector (e.g.,pCR®2.1-TOPO plasmid available from Invitrogen Corp., Carlsbad, Calif.)and the DNA sequence of the Ig H chain variable (V) region (includingthe Vh, D and J segments) and the kappa L chain V-region (including theVk and Jk segments) are determined. The V-region DNA sequences may bedetermined by automated DNA sequencing (DNA sequencing services areavailable from Charles River Laboratories International, Inc.,Wilmington, Mass.).

To isolate the corresponding genomic Ig genes, the genomic DNA isolatedfrom the anti-HCV B cell line (see above) is used as a template for PCRamplification of the human H chain gene and kappa L chain gene. PCRprimers (oligonucleotides) to amplify the V-region genes, (includingtheir respective promoters and flanking regions upstream (i.e., 5′ ofthe V genes) are determined by searching a human genome database withthe V-region DNA sequences established from the cloned Ig mRNA. Forexample a human genome nucleotide database available from the NationalCenter for Biotechnology Information can be searched with a computerprogram, BLAST, for sequences matching the H- and L-chain V-regions. AHuman RefSeq Genome database and BLAST software are available online(see e.g., http://blast.ncbi.nlm.nih.gov/Blast.cgi). Primers to amplifythe Ig constant regions, enhancer sequences, the H-chain membraneanchors, poly A addition sites and downstream flanking regions (i.e., 3′of the Ig gene) are described (see e.g., U.S. Pat. No. 7,741,077 Ibid.).The PCR-amplified, genomic fragments can be cloned in a plasmid vectorsuch as pCR®2.1-TOPO available from Invitrogen Corp., Carlsbad, Calif.).Memory B cells expressing an anti-DNP membrane IgG antibody areengineered to express Ig genes encoding a secreted IgG antibody specificfor HCV. The anti-HCV IgG H chain gene (i.e., γ-H chain gene) may beengineered to remove coding sequences for the membrane spanning domain(TM); the cytoplasmic amino acids (Cyt) and a polyA addition site toyield a γ-H chain gene encoding a secreted H chain only. See FIG. 3 andAbbas et al., Ibid. Ig genes are engineered using standard methods inmolecular biology (see e.g., Sambrook et al., In: Molecular Cloning: ALaboratory Manual, 2^(nd) Ed., Cold Spring Harbor Press, Cold SpringHarbor, N.Y., 1989 which is incorporated herein by reference) to removethe membrane exons and to retain the promoter and enhancer sequencesassociated with the functional anti-HCV Ig genes (see e.g., Abbas etal., Ibid.). The Ig H and L chain genes encoding the antiviral antibodymay be inserted in the non-expressed Ig gene loci by using methods ofhomologous recombination (see e.g., U.S. Pat. No. 5,202,238 Ibid., U.S.Pat. No. 6,570,061 Ibid. and U.S. Pat. No. 6,841,383 Ibid.).

To promote homologous recombination the Ig genes encoding the H chainand L chain for a secreted anti-HCV antibody are cloned in plasmidtargeting vectors to obtain targeted integration in the correspondinggermline Ig loci on chromosomes 14 and 2 respectively. See FIG. 1. Forexample, sequences 5′ of the J_(H) segments upstream from the germlineμ-H chain gene (see FIG. 2: “Maternal Chromosome 14 GermlineConfiguration”) are cloned upstream (5′) of the anti-HCV γ-H chain genein a targeting plasmid, and sequences downstream (3′) of the μ-H chainmembrane anchor exons are cloned downstream (3′) of the γ-H chain geneto promote recombination at the germline H-chain locus on chromosome 14.Methods for construction of targeting vectors containing targetsequences, replacement genes and selectable markers are described (seee.g., U.S. Pat. No. 5,202,238 Ibid., U.S. Pat. No. 6,570,061 Ibid., andU.S. Pat. No. 6,841,383 Ibid.).

Targeting vectors encoding a secreted anti-HCV antibody are used toreplace the nonfunctional, germline μ-H chain gene and the nonfunctionalkappa L chain gene in memory B cells expressing membrane anti-DNP. Thetargeting vector plasmids are linearized by restriction enzyme digestionand transferred by electroporation into the memory B cells followed byselection for the targeting vector plasmids. Methods and reagents forelectroporation of primary mammalian cells are described (see e.g.,“Electroporation Guide” available from BioRad Inc., Hercules, Calif.which is incorporated herein by reference). Memory B cells, followingelectroporation, are cultured in tissue culture media containing drugssuch as G418 and methotrexate to select for selectable marker genes(i.e., neomycin resistance gene and dihydrofolate reductase,respectively) present on the H and L chain targeting vectors. Selectablemarker genes and their use are described (see e.g., U.S. Pat. No.6,841,383 Ibid.). Electroporated memory B cells with resistance to bothG418 and methotrexate are tested for expression of secreted IgG whichbinds HCV. Following transfection and selection of the memory B cells,those cells producing secreted IgG antibodies specific for HCV areidentified using standard immunoassays to assess B cell supernatants(see e.g., Zhang et al., Proc. Natl. Acad. Sci. USA 107: 732-737, 2010which is incorporated herein by reference).

The engineered memory B cells expressing two different antibodies may beactivated in vitro and assayed for proliferation and production of thesecreted anti-HCV antibody. Engineered anti-DNP memory B cells arecultured in vitro with dinitrophenol-human serum albumin (DNP-HSA isavailable from Biosearch Technologies, Novato, Calif.) to activate thecells. For example, memory B cells at about 10⁵ to10⁶ cells/mL arecultured at 37° C. in tissue culture flasks in standard media (e.g.,RPMI 1640 serum-free media available from Sigma-Aldrich Chem. Co., St.Louis, Mo.) which contain approximately 1 μg/ml of DNP-HSA. In addition,memory B cell cultures may include 1 μg/ml of anti-CD40 antibody and 100ng/ml of interleukin-21 (both are available from R&D Systems,Minneapolis, Minn.) to activate the cells and promote antibodyproduction. Methods to activate memory B cells are described (see e.g.,U.S. Pat. No. 7,378,276 Ibid.). To assess activation the cells aretested in a proliferation assay after 3-5 days in culture. Aliquots ofthe culture are supplemented with ³H-thymidine and cultured anadditional 16 hours. ³H-thymidine uptake is measured by using a liquidscintillation counter (see e.g., U.S. Pat. No. 7,378,276 Ibid.).Equivalent cultures of memory B cells incubated without DNP-HSA serve asnegative controls for the proliferation assay. To assess production ofthe anti-HCV antibody by the activated memory B cells, culturesupernatants derived from approximately 3-5 day cultures are tested byenzyme-linked immunosorbent assay (ELISA) to detect and quantify theanti-HCV antibody. Methods to detect and quantify anti-viral antibodieswith ELISA are described (see e.g., Corti et al., Science 333: 850-856,2011 which is incorporated herein by reference). Virions or viralproteins are adsorbed to microtiter plates to capture anti-viralantibodies and a secondary antibody (e.g., anti-IgG) is used to detectthe anti-viral antibodies. Anti-viral antibodies in the concentrationrange of approximately 1 ng/ml to 10,000 ng/ml may be detected using anELISA. A purified anti-HCV antibody produced by a recombinant cell line(see e.g., Wrammert et al., Nature 453: 667-671, 2008 which isincorporated herein by reference) may be used to create standard curvesfor determining antibody concentration in the ELISA assay. Supernatantsfrom engineered memory B cells that are not activated (i.e. culturedwithout DNP-HSA) serve as negative control samples for the anti-HCVantibody ELISA.

To insure the engineered memory B cells are safe for use in patients asuicide gene is introduced in the B cells. To stop uncontrolledproliferation (and/or other adverse events) a suicide gene, Herpessimplex virus-thymidine kinase gene (HSV-TK) is introduced into theengineered memory B cells using a retroviral expression vector. Methodsto insert and express the HSV-TK gene and to activate a cytotoxicprodrug such as ganciclovir are known (see e.g., U.S. Pat. No. 6,576,464issued to Gold and Lebkowski on Jun. 10, 2003 and U.S. Pat. No.5,997,859 issued to Barber et al. on Dec. 7, 1999 which are incorporatedherein by reference). If the engineered B cells are deemed unsafe orcontributing to an adverse event the B cells expressing HSV-TK aretreated with 20 μM ganciclovir (available as Cytovene IV from RocheLaboratories, Nutley, N.J.). Conversion of ganciclovir into a toxicmetabolite by the B cells expressing HSV-TK results in their death.Cells not expressing HSV-TK are not harmed by ganciclovir.

Engineered memory B cells expressing an anti-DNP BCR and an anti-viral(anti-HCV) secreted antibody may be expanded and used for adoptive celltherapy of the patient with chronic HCV infection. The B cells may beactivated in vitro (as described above) or in vivo by administration ofDNP-HSA to the patient. Immunization with approximately 100 mg DNP-KLHadministered subcutaneously may be done to activate the engineeredmemory B cells (see e.g., Rentenaar et al., Ibid.). Multiple activationsmay be stimulated to respond to HCV infections.

Example 3 Mature B Lymphocytes Engineered to Express a Membrane AntibodySpecific for Prostate Specific Antigen and a Second, Secreted AntibodySpecific for Prostate Cancer Lipid Antigen

An isolated recombinant B lymphocyte cell line that produces a secretedimmunoglobulin against prostate cancer lipid antigen (PCLA) and producesa membrane immunoglobulin to prostate specific antigen (PSA) can beutilized for cell therapy to treat prostate cancer in a mammaliansubject. The recombinant B lymphocyte cell line can be injected into themammalian subject as adoptive cell therapy to provide immunologicalreactivity to PSA on prostate cancer cells and to process and presentPSA to T lymphocytes. The recombinant B lymphocyte cell line can beactivated by endogenous PSA arising in the subject to produce secretedanti-PCLA antibody. The recombinant B lymphocyte cell line can also beactivated in vivo or ex vivo by injecting the mammalian subject (or anin vitro cell culture) with exogenous prostate specific antigen (PSA) toproduce secreted anti-PCLA antibody. Determination of timing tostimulate immunological reactivity to prostate cancer cells in themammalian subject can be chosen based upon the detection of prostatecancer cells in the mammalian subject.

Polyclonal mature B cells expressing B cell receptors (BCR) comprised ofmembrane IgM and IgD are isolated from a prostate cancer patient. MatureB cells may be obtained from peripheral blood leukocytes of the patient.For example approximately 10⁹ leukocytes may be harvested using aleukapheresis procedure (see e.g., Bensinger et al., Blood 81:3158-3163, 1993 which is incorporated herein by reference) andapproximately 5% (i.e., 5×10⁷ cells) are B cells. Mature B cells areisolated from the patient's leukocytes by using antibodies specific forB cell markers CD19, IgD, CD38, and CD21 (available from BectonDickinson/Pharmingen, San Diego, Calif.). Methods to purify mature Bcells using magnetic beads (available from Miltenyi Biotech, Auburn,Calif.) and a fluorescence-activated cell sorter (FACS) are described(see e.g., U.S. Pat. No. 7,378,276 Ibid.). Mature B cells expressingmembrane IgM and IgD are cultured in vitro and genetically engineered toexpress two different antibodies.

Mature B cells are genetically engineered to express a membrane IgGantibody specific for prostate specific antigen (PSA). PSA is a proteinantigen associated with prostate cancer that may be produced usingrecombinant DNA methods and purified for use as an antigen (see e.g.,U.S. Pat. No. 8,013,128 issued to Gudas et al. on Sep. 6, 2011 which isincorporated herein by reference). To obtain human immunoglobulin (Ig)genes encoding an antibody specific for PSA a hybridoma cell line thatproduces the anti-PSA antibody is constructed. For example transgenicmice with human Ig genes (e.g., XenoMouse® available from Abgenix Inc.,Fremont, Calif.) are immunized with PSA and their B cells are fused witha myeloma cell fusion partner, e.g. SP2/0 cells (available from AmericanType Culture Collection, Manassas, Va.) to create hybridoma cell clonesexpressing human antibodies (see e.g., U.S. Pat. No. 8,013,128 Ibid.).Supernatants from the hybrid clones are screened using an immunoassay todetect human IgG antibodies which bind PSA protein. Hybridoma clonesproducing antibodies that recognize PSA are expanded and antibodies fromeach clone are tested using a Biacore™ A100 instrument (available fromGE Healthcare, Piscataway, N.J.) to measure antibody affinity andspecificity for PSA (see e.g., GE Healthcare, Application Note 84,“Early kinetic screening of hybridomas . . . ”, which is incorporatedherein by reference). Hybridomas expressing high affinity antibodies forPSA are selected for cloning of their human Ig genes. Methods to cloneIg heavy (H) chain and light (L) chain genes may be used. See e.g., U.S.Pat. No. 7,741,077 issued to Grawunder et al. on Jun. 22, 2010 and Earlyet al., Proc. Natl. Acad. Sci. USA 76: 857-861, 1979 which areincorporated herein by reference. For example, a hybridoma cell lineexpressing a human anti-PSA antibody, IgG₁(kappa), is grown in cultureand used as a source to isolate messenger RNA (mRNA) and genomic DNAusing standard methods employing phenol/chloroform (see e.g., Sambrooket al., In: Molecular Cloning: A Laboratory Manual, 2^(nd) Ed., ColdSpring Harbor Press, Cold Spring Harbor, N.Y., 1989). The mRNA encodingthe IgG₁ H-chain and the kappa L-chain are molecularly cloned followingamplification using the polymerase chain reaction (PCR) and reversetranscriptase (RT). Methods and Ig gene primers to amplify the H chainand L chain mRNA are described in U.S. Pat. No. 7,741,077 Ibid. The Hand L chain mRNA (amplified as complementary DNA) are cloned in aplasmid vector (e.g., pCR®2.1-TOPO plasmid available from InvitrogenCorp., Carlsbad, Calif.) and the DNA sequence of the Ig H chain variable(V) region (including the Vh, D and J segments) and the kappa L chainV-region (including the Vk and Jk segments) are determined. The V-regionDNA sequences may be determined by automated DNA sequencing (DNAsequencing services are available from Charles River LaboratoriesInternational, Inc., Wilmington, Mass.). To isolate the correspondinggenomic Ig genes, the genomic DNA isolated from the anti-PSA hybridoma(see above) is used as a template for PCR amplification of the human Hchain gene and kappa L chain gene. PCR primers (oligonucleotides) toamplify the V-region genes, (including their respective promoters andflanking regions upstream (i.e., 5′ of the V genes) are determined bysearching a human genome database with the V-region DNA sequencesestablished from the cloned Ig mRNA. For example a human genomenucleotide database available from the National Center for BiotechnologyInformation can be searched with a computer program, BLAST, forsequences matching the H- and L-chain V-regions. A Human RefSeq Genomedatabase and BLAST software are available online (see e.g., the worldwide web at blast.ncbi.nlm.nih.gov/Blast.cgi). Primers to amplify the Igconstant regions, enhancer sequences, the H-chain membrane anchors, polyA addition sites and downstream flanking regions (i.e., 3′ of the Iggene) are described (see e.g., U.S. Pat. No. 7,741,077 Ibid.). ThePCR-amplified, genomic fragments can be cloned in a plasmid vector suchas pCR®2.1-TOPO available from Invitrogen Corp., Carlsbad, Calif.). TheIgG₁ H chain gene (i.e., γ₁-H chain gene) may be engineered to removethe “tail piece” and polyadenylation site encoding the secreted form ofthe H chain, thus only a membrane γ₁-H chain is encoded by theengineered gene (see e.g., FIG. 3, and Abbas et al., Cellular andMolecular Immunology, 7^(th)Ed., Elsevier Saunders, Philadelphia, Pa.,2011 which is incorporated herein by reference). For example, the clonedγ₁-H chain gene may be amplified by PCR with primers that amplify theγ₁-H chain constant region gene but omit the tail piece andpolyadenlyation site encoding the secreted form of the γ₁-H chain. Theprimer may also add a RNA splice donor site to the 3′ end of the γ₁-Hchain gene and a unique restriction enzyme site (e.g., a site for Not I;enzyme available from New England Biolabs, Ipswich, Mass.). Separate DNAfragments encoding the membrane anchor exons and the remainder of theγ₁-H chain gene are PCR-amplified using PCR primers containingrestriction enzyme sites which allow reassembly of the γ₁-H geneencoding a membrane form of the γ₁-H chain. See FIG. 3. Methods toamplify and assemble Ig genes are described (see e.g., U.S. Pat. No.7,741,077 Ibid.).

The Ig genes encoding the H chain and L chain for an anti-PSA membraneantibody are cloned in plasmid targeting vectors to obtain targetedintegration in the corresponding nonfunctional germline Ig loci onchromosomes 14 and 2 respectively. See FIG. 1. For example, sequences 5′of the J_(H) genes (see FIG. 2A) are cloned upstream (5′) of theanti-PSA γ₁-H chain gene in a targeting plasmid, and sequencesdownstream (3′) of the μ-H chain membrane anchor exons (TM and Cyt) arecloned downstream (3′) of the γ₁-H chain gene to promote recombinationat the germline H-chain locus on chromosome 14. Methods for constructionof targeting vectors containing target sequences, replacement genes andselectable markers are described (see e.g., U.S. Pat. No. 5,202,238issued to Perry et al. on Apr. 13, 1993; U.S. Pat. No. 6,570,061 issuedto Rajewsky and Zou on May 27, 2003, and U.S. Pat. No. 6,841,383 issuedto Reff et al. on Jan. 11, 2005 which are incorporated herein byreference). Targeting vectors constructed to replace the nonfunctionalgermline chain gene and the nonfunctional kappa L chain gene in mature Bcells are transferred into mature B cells in vitro. The targeting vectorplasmids are linearized by restriction enzyme injection and transferredby electroporation into the mature B cells followed by selection for thetargeting vector plasmids. Methods and reagents for electroporation ofprimary mammalian cells are described (see e.g., “Electroporation Guide”available from BioRad Inc., Hercules, Calif. which is incorporatedherein by reference). Mature B cells, following electroporation arecultured in tissue culture media containing drugs such as G418 andmethotrexate to select for selectable marker genes (i.e., neomycinresistance gene and dihydrofolate reductase, respectively) present onthe H and L chain targeting vectors. Selectable marker genes and theiruse are described (see e.g., U.S. Pat. No. 6,841,383 Ibid.)Electroporated mature B cells with resistance to both G418 andmethotrexate are tested for expression of membrane IgG which binds PSA.For example, engineered mature B cells expressing membrane IgG specificfor PSA are isolated using magnetic beads with PSA attached, and thecells are propagated in vitro prior to transfection with Ig genes for asecond antibody specific for a different prostate tumor associatedantigen.

Mature B cells expressing an anti-PSA membrane IgG antibody areengineered to express Ig genes encoding a secreted IgG antibody specificfor prostate cancer lipid antigen (PCLA). Methods to extract PCLA and toobtain a monoclonal antibody specific for PCLA are known (see e.g.,Zhang et al., Proc. Natl. Acad. Sci. USA 107: 732-737, 2010 which isincorporated herein by reference). A human IgG antibody specific forPCLA and the corresponding Ig genes may be obtained as described above(see e.g., U.S. Pat. No. 7,741,077 Ibid. and Early et al., Ibid.). Theanti-PCLA IgG H chain gene (i.e., γ-H chain gene) may be engineered toremove coding sequences for the membrane spanning domain and thecytoplasmic amino acids to yield a γ-H chain gene encoding a secreted Hchain only. See FIG. 3 and Abbas et al., Ibid. The anti-PCLA Ig genesare integrated into the functionally rearranged Ig gene loci of themature B cell which include the μ-H chain gene on chromosome 14 and thekappa L chain gene on chromosome 2 (e.g., see FIG. 2; only the H chaingene is shown). Targeted integration of the anti-PCLA γ-H chain gene andL chain gene into the corresponding functional H and L chain gene loci(i.e. chromosomes 14 and 2 respectively) is done using methods ofhomologous recombination as described above (see U.S. Pat. No. 6,570,061Ibid., and U.S. Pat. No. 6,841,383 Ibid.). To target integration intothe functional μ-H chain locus, targeting sequences from the intronbetween the J_(H) cluster and the μ constant region gene (CHO are placed5′ of the anti-PCLA γ-H chain gene and sequences downstream from the μmembrane anchor exons are placed 3′ of the γ-H chain gene (see FIG. 2).Analogous targeting sequences (i.e., from the Jk-Ck intron and 3′ of theCk gene) are used for targeting the anti-PCLA kappa light chain gene tothe functional Ck gene. The targeting vectors for anti-PCLA H and Lchain include different selectable marker genes, hygromycin resistanceand histidinol dehydrogenase, respectively. Media containing hygromycinand histidinol is used to select for engineered mature B cellsexpressing secreted IgG anti-PCLA antibody. Essential transcriptionalpromoter sequences and enhancer sequences necessary for Ig geneexpression are retained in the Ig gene integrants (see Abbas et al.,Ibid.). Following transfection and selection of the mature B cells,those cells producing secreted IgG antibodies specific for PCLA areidentified using standard immunoassays to assess B cell supernatants(see e.g., Zhang et al., Ibid.). The engineered mature B cells arecultured in vitro and stimulated with PSA to activate the cells and tostimulate secretion of anti-PCLA IgG antibodies.

To insure the engineered mature B cells are safe for use in patients asuicide gene is introduced in the B cells. To stop uncontrolledproliferation (and/or other adverse events) a suicide gene, Herpessimplex virus-thymidine kinase gene (HSV-TK) is introduced into theengineered memory B cells using a retroviral expression vector. Methodsto insert and express the HSV-TK gene and to activate a cytotoxicprodrug such as ganciclovir are known (see e.g., U.S. Pat. No. 6,576,464issued to Gold and Lebkowski on Jun. 10, 2003 and U.S. Pat. No.5,997,859 issued to Barber et al. on Dec. 7, 1999 which are incorporatedherein by reference). If the engineered B cells are deemed unsafe orcontributing to an adverse event the B cells expressing HSV-TK aretreated with 20 μM ganciclovir (available as Cytovene IV from RocheLaboratories, Nutley, N.J.). Conversion of ganciclovir into a toxicmetabolite by the B cells expressing HSV-TK results in their death.Cells not expressing HSV-TK are not harmed by ganciclovir.

The isolated recombinant B lymphocytes are administered to prostatecancer patients to provide antibodies to PCLA and to process and presentPSA to T cells. Autologous B cells engineered to express anti-PSAmembrane IgG and anti-PCLA secreted IgG are cultured in vitro withapproximately 1 μg/mL PSA for approximately 3 to 5 days and then washedin serum-free media prior to injection. Approximately 5-10×10⁸ B cellsare injected intravenously and the concentration of anti-PCLA antibodiesand the number of engineered B cells in the peripheral blood of thepatient are monitored with immunoassays and flow cytometry respectively.

Example 4 Memory B Lymphocytes from Patients Vaccinated with InfluenzaVaccine are Provided with Membrane Antibodies Specific for DNP andActivated by Administration of DNP-HSA

An isolated recombinant B lymphocyte cell line that produces a secretedbroadly neutralizing immunoglobulin to influenza virus and produces amembrane immunoglobulin to a model antigen can be utilized for celltherapy in a mammalian subject. The recombinant B lymphocyte cell linecan be injected into the mammalian subject as cell therapy to provideimmunological protection from infection by influenza virus. Therecombinant B lymphocyte cell line can be activated in vivo or ex vivoto produce the broadly neutralizing influenza antibody by injecting themammalian subject (or an in vitro cell culture) with model antigen,dinitrophenol-keyhole limpet hemocyanin (DNP-KLH). The timing tostimulate immunological protection from influenza virus infection in themammalian subject can be chosen based upon the timing of an outbreak ofinfluenza infection in the population at large.

An individual is immunized with an influenza vaccine to obtain memory Bcells with B cell receptors (BCR) specific for influenza virus. Memory Bcells develop in response to immunization with a subunit vaccine forinfluenza virus which may elicit broadly neutralizing antibodies (seee.g., Ekiert et al., Science 324: 246-251, 2009 which is incorporatedherein by reference). A primary immunization with 1 mg of influenzavirus vaccine, for example, a conserved epitope from the viralhemagglutinin (HA) protein is injected subcutaneously in the right arm.Approximately 12-14 days after immunization memory B cells expressingBCR specific for influenza are isolated using influenza HAprotein-biotin and phycoerythrin (PE)-streptavidin and afluorescein-anti-CD27 antibody to identify memory B cells (biotin,streptavidin and antibodies are available from BectonDickinson/Pharmingen, San Diego, Calif.). Influenza-specific memory Bcells are isolated by cell sorting with a fluorescence activated cellsorter (e.g., FACSAriaIII® available from Becton Dickinson, FranklinLakes, N.J.). For example, see U.S. Pat. No. 7,378,276 issued toEttinger et al. on May 27, 2008 and U.S. Pat. No. 7,993,864 issued toBrown et al. on Aug. 9, 2011 which are incorporated herein by reference.Memory B cells expressing membrane IgG specific for an influenza HAepitope are cultured in vitro and expanded prior to transfection with amembrane immunoglobulin specific for dinitrophenol (DNP). For example,memory B cells at about 10⁵ to10⁶ cells/mL are cultured at 37° C. intissue culture flasks in standard media (e.g., RPMI 1640 serum-freemedia available from Sigma-Aldrich Chem. Co., St. Louis, Mo.) whichcontain approximately 1 μg/ml of influenza HA peptide (see e.g., Ekiertet al., Ibid.) In addition, memory B cell cultures may include 1 μg/mlof anti-CD40 antibody and 100 ng/ml of interleukin-21 (both areavailable from R&D Systems, Minneapolis, Minn.) to activate the cells(see e.g., U.S. Pat. No. 7,378,276 Ibid.). A membrane immunoglobulinspecific for DNP is produced using recombinant DNA methods and insertedin the membrane of memory B cells producing anti-influenza antibodies.Immunoglobulin (Ig) genes encoding a membrane IgG antibody specific forDNP may be obtained from healthy volunteers who are immunized withDNP-KLH (see e.g., Biosearch Technologies DNP-KLH Product Info Sheetwhich is incorporated herein by reference). Memory B cells with membraneIgG recognizing DNP are isolated by cell sorting with afluorescence-activated cell sorter (e.g., FACSAriaIII® available fromBecton Dickinson, Franklin Lakes, N.J.). For example, see U.S. Pat. No.7,378,276 Ibid. and U.S. Pat. No. 7,993,864 Ibid.

Immunoglobulin genes encoding an anti-DNP antibody are isolated fromindividual B cells (see e.g., Tiller et al., J. Immunol. Methods 329:112-124, 2008 which is incorporated herein by reference). For eachindividual anti-DNP B cell the Ig heavy (H) and corresponding Ig light(L) chain gene transcripts are amplified by reversetranscriptase-polymerase chain reaction (RT-PCR) using Superscript®IIIreverse transcriptase (available from Invitrogen Corp., Carlsbad,Calif.) and Taq DNA polymerase (available from Qiagen, Valencia,Calif.). Reaction conditions and oligonucleotide primers to amplify Ig Hchains and Ig L chains are known (see e.g. Tiller et al., Ibid.). TheDNA fragments encoding the Ig H and Ig L chain variable (V) region genesare isolated and cloned in mammalian expression vectors containing Ig Hand Ig L chain constant region genes (e.g., Cγ1 and Ck). DNA sequencesof the cloned anti-DNP Ig genes (γ1-H chain and κ-L chain) aredetermined using a DNA sequencer (e.g., using a 3130 Genetic Analyzeravailable from Applied Biosystems, Carlsbad, Calif.). The IgG₁ H chaingene (i.e., γ₁-H chain gene) is engineered to remove the “tail piece”and polyadenylation site encoding the secreted form of the H chain, thusonly a membrane γ₁-H chain is encoded by the engineered gene (see e.g.,FIG. 3B, and Abbas et al., Cellular and Molecular Immunology, 7th Ed.,Elsevier Saunders, Philadelphia, Pa., 2012 which is incorporated hereinby reference). For example, the cloned γ₁-H chain gene may be amplifiedby PCR with primers that amplify the γ₁-H chain constant region gene butomit the tail piece and polyadenlyation site encoding the secreted formof the γ₁-H chain. A separate DNA fragment encoding the γ₁ membraneanchor exons, and the remainder of the γ₁-H chain gene are PCR-amplifiedusing PCR primers containing restriction enzyme sites which allowreassembly of the γ₁-H gene encoding a membrane form of the γ₁-H chain.See FIG. 3B. Methods to amplify and assemble Ig genes are described (seee.g., U.S. Pat. No. 7,741,077 Ibid.).

The genetically engineered immunoglobulin genes encoding the anti-DNPmembrane antibody are expressed in a mammalian cell line and themembrane IgG is purified from the cell line. For example, a kappa (κ) Lchain gene and the modified γ-1 H chain gene are inserted in alentiviral expression vector using standard recombinant DNA methods (seee.g., U.S. Patent Publication No. 2007/0116690 by Yang et al. publishedon May 24, 2007 which is incorporated herein by reference). The viralvector is used to transfect Chinese Hamster Ovary (CHO) cells (availablefrom American Type Culture Collection, Manassas, Va.) which areengineered to express membrane immunoglobulin. Methods to expressmembrane immunoglobulins may be used. See e.g., Price et al., J Immunol.Methods 343: 28-41, 2009 which is incorporated herein by reference. Toidentify and isolate CHO clones expressing the anti-DNP membrane IgG aphycoerythrin-conjugated anti-human IgG antibody was used to label CHOcells and sort them using FACS (see e.g., Price et al., Ibid.). A CHOcell line producing anti-DNP membrane IgG is isolated and expanded andmembrane IgG is purified from CHO cell lysates using an immunoaffinitycolumn. An affinity column constructed from protein A-Sepharose(available from Sigma-Aldrich Co., St. Louis, Mo.) is used to purifymembrane IgG from lysates of the engineered CHO cells. For example cellsmay be lysed in a buffer containing: 0.15 M NaCl, 0.01 M TrisHCl, pH8.2, 1 mM EDTA, 2 mM phenylmethylsulfonyl fluoride, 0.5% Nonidet P-40and 1 mg/mL HSA (see e.g., Schneider et al., J. Biol. Chem. 257:10766-10769, 1982 which is incorporated herein by reference). Thepurified anti-DNP membrane IgG is used to construct liposomes which arefused to memory B cells specific for influenza antigen (see above).

Liposomes containing anti-DNP membrane IgG are constructed fromphospholipids and purified anti-DNP membrane IgG antibodies. Theliposomes with incorporated anti-DNP membrane IgG antibodies may befused with memory B cells specific for influenza virus to obtain memoryB cells with anti-DNP membrane immunoglobulin incorporated into the Bcell membrane. Liposomes may be prepared from cholesterol andL-α-phosphatidylcholine. See e.g., U.S. Patent Publication No.2005/0208120, which is incorporated herein by reference. Cholesterol andL-α-phosphatidyl choline are combined at a molar ratio of 2:7 inchloroform and the chloroform is evaporated away using an argon stream.The liposomes are resuspended in a 140 mM NaCl, 10 mM Tris HCl, 0.5%deoxycholate at pH 8 and sonicated for three minutes. Purified anti-DNPmembrane antibodies (see above) are inserted into the liposomes bycombining the membrane IgG with liposomes at a 1:10 molar ratio anddialyzing for 72 hours at 4° C. versus phosphate buffered saline. Theliposomes are characterized to assess liposome size and the amount ofanti-DNP membrane IgG protein incorporated into the liposomes. Liposomesize is determined using dynamic light scattering and flow cytometry(see e.g., U.S. Patent Application No. 2005/0208120 by Albani which isincorporated herein by reference). For example, liposomes containinganti-DNP antibodies may have a mean diameter of approximately 50nanometers. To measure anti-DNP IgG protein on the liposomes theliposomes are analyzed on a flow cytometer after staining withFITC-labeled anti-IgG antibody. Liposomes are sorted based on FITCfluorescence, forward scatter and side scatter to isolate and countliposomes with IgG. Anti-DNP IgG protein on the liposomes is measuredusing an enzyme-linked immunosorbent assay (ELISA). Methods to analyzeliposomes by flow cytometry and to measure IgG and other proteins byELISA are known (see e.g., U.S. Patent Application No. 2005/0208120,Ibid.).

Liposomes containing anti-DNP membrane IgG are fused with memory B cellsspecific for influenza virus (see above) to obtain memory B cells withanti-DNP B cell receptor. Purified liposomes with anti-DNP BCR on theirsurface are electrofused with the memory B cells (see e.g., Zimmermannet al., IEEE Transactions On Plasma Science 28: 72-82, 2000 which isincorporated herein by reference). For example a 1:1 ratio of liposomesto memory B cells are suspended in a hypo-osmolar buffer containing 0.1mM Ca-acetate, 0.5 mM Mg-acetate and 1 mg/ml bovine serum albumin. Theosmolarity is adjusted to approximately 75 mOsm and approximately 200 μLof the cell suspension containing approximately 2×10⁴ to 2×10⁵ cells isplaced in an electrofusion chamber (electrofusion generators andchambers are available from BTX Instrument Division, Harvard Apparatus,Inc. Holliston, Mass.). The cells are aligned by applying an alternatingfield of 5 V amplitude and 2 MHz frequency for approximately 30 seconds.Then fusion is initiated by applying a rectangular fusion pulse of 20Vto 40V amplitude and of 15 μsec duration. The alternating field isapplied again for 30 seconds to maintain cells and liposomes in positionwhile fusion occurs. The cells are transferred to culture flasks andgrown for 2 to 5 weeks.

The fused memory B cells are characterized to assess their anti-DNP BCRsand their production of anti-influenza antibodies. The fused memory Bcells are tested for membrane anti-DNP antibodies using fluorescentDNP-HSA and FACS analysis. Methods to assess membrane anti-DNP IgGantibodies using flow cytometry are described above (see Example 2). Thefused memory B cells may be activated in vitro to produce anti-influenzaantibodies when stimulated with DNP-HSA, and the production of secretedanti-influenza virus antibodies may be measured using an ELISA basedupon influenza viral hemagglutinin protein or influenza virions. Methodsto measure anti-influenza antibodies and memory B cell activation areknown (see e.g., U.S. Pat. No. 7,378,276 Ibid. and Example 1).

The human patient at risk of influenza viral infection is givenrecombinant fused memory B cells as a therapeutic and prophylactic celltherapy which can be activated in vivo. The recombinant memory B cellsare activated in vivo by administration of DNP-HSA to the patient whenan anti-influenza antibody response is needed. For example,approximately 10⁸-10⁹ fused B cells may be injected as a prophylacticwhen the patient is healthy prior to “flu season.” The fused memory Bcells may be activated when needed by intracutaneous injection of 100 μgof DNP-HSA to the patient. For example, the fused memory B cells may beactivated after the patient is exposed to influenza virus or at thefirst signs of infection. The production of anti-influenza antibodiesmay be monitored by sampling the patient's peripheral blood andperforming ELISA with influenza virions as the antigen. Moreover, thepresence of broadly neutralizing antibodies for multiple strains ofinfluenza virus can be determined by ELISA based on conserved epitopesfrom influenza virus (see e.g., Ekiert et al., Ibid.)

Example 5 Construction of Autologous Memory B Lymphocytes Engineered toProduce Two Different Anti-Staphylococcus aureus Antibodies

An isolated recombinant B lymphocyte cell line that produces twodifferent secreted immunoglobulins to methicillin-resistantStaphylococcus aureus (MRSA) and produces a membrane immunoglobulin to athird S. aureus antigen can be utilized for cell therapy in a mammaliansubject. The recombinant B lymphocyte cell line can be injected into themammalian subject as cell therapy to provide immunological protectionfrom infection by MRSA. The recombinant B lymphocyte cell line can beactivated in vivo or ex vivo to produce antibody to MRSA by injectingthe mammalian subject (or an in vitro cell culture) with S. aureusantigen. The timing to stimulate immunological protection from MRSAinfection in the mammalian subject can be chosen based upon the exposureof the subject to MRSA or the appearance of symptoms of MRSA infection.

A patient infected with methicillin-resistant Staphylococcus aureus(MRSA) who has suffered recurring episodes of infection is treated withhis own long-lived, memory B cells which have been geneticallyengineered to express two different anti-S. Aureus monoclonal antibodies(MAb). Memory B cells expressing membrane IgG (also known as surface IgGor B cell receptor (BCR)) are isolated from the peripheral blood of thepatient with a recurrent MRSA infection. Polyclonal memory B cells withunknown antigen specificities are isolated from the patient's peripheralblood: 1) by isolating peripheral blood mononuclear cells using FicollHypaque density gradients (available from Sigma Aldrich, St. Louis,Mo.); 2) by negative selection of total B cells using magnetic beads(available from Stem Cell Technology, Vancouver, BC), and 3) by labelingthe cells with fluorescent monoclonal antibodies that recognize IgG andCD27, a memory B cell marker, and performing fluorescence-activated cellsorting. See for example, U.S. Pat. No. 7,378,276 issued to Ettinger etal. on May 27, 2008 and U.S. Pat. No. 7,993,864 issued to Brown et al.on Aug. 9, 2011 which are incorporated herein by reference. The purifiedmemory B cells are modified using genetic engineering methods tointroduce immunoglobulin (Ig) genes encoding two different anti-S.aureus antibodies.

Ig genes encoding a first anti-S. aureus IgG antibody are isolated froma hybridoma cell line which produces the antibody. Methods to constructa hybridoma cell line producing an IgG antibody specific forpoly-N-acetylglucosamine (PNAG) which is protective against S. aureusare described (see e.g., Kelly-Quintos et al., Infection and Immunity74: 2742-2750, 2006 which is incorporated herein by reference). Forexample transgenic mice with human Ig genes (e.g., XenoMouse® availablefrom Abgenix Inc., Fremont, Calif.) are immunized with PNAG and their Bcells are fused with a myeloma cell fusion partner, e.g. SP2/0 cells(available from American Type Culture Collection, Manassas, Va.) tocreate hybridoma cell clones expressing human antibodies (see e.g., U.S.Pat. No. 8,013,128 Ibid.) Hybridomas expressing high affinity antibodiesfor PNAG are selected for cloning of their Ig genes. Methods to clone Igheavy (H) chain and light (L) chain genes are known (see e.g., U.S. Pat.No. 7,741,077 issued to Grawunder et al. on Jun. 22, 2010 and Early etal., Proc. Natl. Acad. Sci. USA 76: 857-861, 1979 which are incorporatedherein by reference). For example, a hybridoma cell line expressing ananti-PNAG antibody, IgG₁(kappa), is grown in culture and used as asource to isolate messenger RNA (mRNA) and genomic DNA using standardmethods employing phenol/chloroform (see e.g., Sambrook et al., In:Molecular Cloning: A Laboratory Manual, 2^(nd) Ed., Cold Spring HarborPress, Cold Spring Harbor, N.Y., 1989). The mRNA encoding the IgG₁H-chain and the kappa L-chain are molecularly cloned followingamplification using the polymerase chain reaction (PCR) and reversetranscriptase (RT). Methods and Ig gene primers to amplify the H chainand L chain mRNA are described in U.S. Pat. No. 7,741,077 Ibid. The Hand L chain mRNA (amplified as complementary DNA) are cloned in aplasmid vector (e.g., pCR®2.1-TOPO plasmid available from InvitrogenCorp., Carlsbad, Calif.) and the DNA sequence of the Ig H chain variable(V) region (including the Vh, D and J segments) and the kappa L chainV-region (including the Vk and Jk segments) are determined. The V-regionDNA sequences may be determined by automated DNA sequencing (DNAsequencing services are available from Charles River LaboratoriesInternational, Inc., Wilmington, Mass.).

To isolate the corresponding genomic Ig genes, the genomic DNA isolatedfrom the anti-PNAG hybridoma, as isolated above, is used as a templatefor PCR amplification of the H chain gene and kappa L chain gene. PCRprimers (oligonucleotides) to amplify the V-region genes, (includingtheir respective promoters and flanking regions upstream (i.e., 5′ ofthe V genes) are determined by searching a human genome database withthe V-region DNA sequences established from the cloned Ig mRNA. Forexample a human genome nucleotide database available from the NationalCenter for Biotechnology Information can be searched with a computerprogram, BLAST, for sequences matching the H- and L-chain V-regions. AHuman RefSeq Genome database and BLAST software are available online(see e.g., the world wide web at blast.ncbi.nlm.nih/gov/blast.cgi).Primers to amplify the Ig constant regions, enhancer sequences, theH-chain membrane anchors, poly A addition sites and downstream flankingregions (i.e., 3′ of the Ig gene) are described (see e.g., U.S. Pat. No.7,741,077 Ibid.). The PCR-amplified, genomic fragments can be cloned ina plasmid vector such as pCR®2.1-TOPO available from Invitrogen Corp.,Carlsbad, Calif.). The IgG₁ H chain gene (i.e., γ₁-H chain gene) may beengineered to remove the “tail piece” and polyadenylation site encodingthe secreted form of the H chain, thus only a membrane γ₁-H chain isencoded by the engineered gene (see e.g., FIG. 3B, and Abbas et al.,Cellular and Molecular Immunology, 7^(th) Ed., Elsevier Saunders,Philadelphia, Pa., 2011 which is incorporated herein by reference). Forexample, the cloned γ₁-H chain gene may be amplified by PCR with primersthat amplify the γ₁-H chain constant region gene but omit the tail pieceand polyadenlyation site encoding the secreted form of the γ₁-H chain.Separate DNA fragments encoding the membrane anchor exons and theremainder of the γ₁-H chain gene are PCR-amplified using PCR primerscontaining restriction enzyme sites which allow reassembly of the γ₁-Hgene encoding a membrane form of the γ₁-H chain. See FIG. 3B. Methods toamplify and assemble Ig H and L chain genes are described (see e.g.,U.S. Pat. No. 7,741,077 Ibid.).

The Ig genes encoding the heavy (H) chain and light (L) chain of theanti-PNAG antibody are cloned in targeting plasmid vectors to allowtargeted integration at and replacement of the correspondingfunctionally rearranged Ig H and Ig L chain genes on chromosomes 14 and2 respectively (e.g., See FIG. 1). Methods to target genes to Ig lociusing homologous recombination are known (see e.g., U.S. Pat. No.5,202,238 issued to Perry et al. on Apr. 13, 1993; U.S. Pat. No.6,570,061 issued to Rajewsky and Zou on May 27, 2003 and U.S. Pat. No.6,841,383 issued to Reff et al. on Jan. 11, 2005 which are incorporatedherein by reference). To target integration into the functional γ₁-Hchain locus, targeting sequences from the intron between the J_(H)cluster and the μ constant region gene (C_(H)μ; see FIG. 2A) are placed5′ of the anti-PNAG γ-H chain gene and sequences downstream from the γ1membrane anchor exons are placed 3′ of the γ-H chain gene (see FIG. 3A).Analogous targeting sequences (i.e., from the J_(K)-C_(K) intron and 3′of the C_(K) gene) are used for targeting the anti-PNAG kappa lightchain gene into the functional C_(K) gene. The targeting vectors foranti-PNAG H and L chain include selectable marker genes, e.g.,hygromycin resistance and Zeocin™ bleomycin resistance, respectively.Media containing hygromycin B and Zeocin™ bleomycin are used to selectfor engineered memory B cells expressing membrane IgG anti-PNAG antibody(Protocols, selection agents and selectable markers are available fromInvitrogen, Carlsbad, Calif.). Essential transcriptional promotersequences and enhancer sequences necessary for Ig gene expression areretained in the Ig replacement genes (see Abbas et al., Ibid.).Following transfection, homologous recombination and selection,recombinant memory B cells expressing membrane IgG specific for S.aureus PNAG are isolated using magnetic beads with PNAG attached(magnetic beads and protocols are available from Miltenyi Biotech Inc.,Auburn, Calif.). Memory B cells expressing the anti-PNAG membraneantibody are cultured in vitro prior to transfection with Ig genesencoding a second antibody specific for a different S. aureus antigen.

Memory B cells are produced using recombinant DNA methods to express amembrane antibody specific for PNAG and are further geneticallyengineered to express a second anti-S. aureus MAb. A monoclonal antibody(MAb) specific for the immunodominant staphylococcal antigen A (IsaA) isexpressed in the memory B cells. To obtain an anti-IsaA MAb withprophylactic and therapeutic activity for S. aureus infections ahybridoma cell line is constructed using purified recombinant IsaAprotein to immunize mice and select hybridoma clones (see e.g., Lorenzet al., Antimicrob. Agents Chemoth. 55: 165-173, 2011 which isincorporated herein by reference). To clone anti-IsaA antibody variableregion genes messenger RNA is extracted from a selected hybridoma cellline and used as a template for complementary DNA synthesis with reversetranscriptase (RT) and amplification using polymerase chain reaction(PCR) (i.e., RT-PCR). Methods to amplify and clone the variable regionsfor heavy and light chain in an antibody expression vector are described(see e.g., Kelly-Quintos et al., Ibid.) For example, a plasmidexpression vector encoding a complete gamma (γ)-1 H chain and a lambda(λ) L chain may be constructed using restriction enzymes and standardmolecular biology methods (see e.g., Sambrook et al., Ibid.). Tofacilitate transfection and expression of the anti-IsaA antibody inmemory B cells the γ1-H chain gene and the λ-L chain gene may betransferred to a lentiviral vector (see e.g., U.S. Pat. No. 7,939,059issued to Yang et al. on May 10, 2011 which is incorporated herein byreference). Infection of memory B cells with the recombinant lentivirusresults in integration of the vector sequences at random sites (i.e.,not targeted) in the genomic DNA of the memory B cells and production ofthe secreted IgG1 (λ) anti-IsaA antibody. Protocols and lentiviralexpression vectors are available from Invitrogen Corp., Carlsbad,Calif.; see e.g., User Manual: “ViraPower™ HiPerfom™ LentiviralExpression System” which is incorporated herein by reference. Forexample a flask of memory B cells is infected with titered recombinantlentivirus stock so as to yield a multiplicity of infection ofapproximately 1.0 transducing units per cell. The cells and lentivirusare incubated overnight at 37° C. in 5% CO₂; then, the lentiviruscontaining media is replaced by fresh media and incubated overnight. Onthe third day the cells are placed in selective media (e.g., mediacontaining blasticidin is available from Invitrogen Corp., Carlsbad,Calif.) to select for stably transduced cells containing the lentiviralvector. Clones of memory B cells resistant to blasticidin are alsoplaced under selection for hygromycin B and Zeocin™ to select clonesexpressing both anti-MRSA antibodies. To identify and purify memory Bcells expressing both anti-S. aureus antibodies memory B cells withsurface IgG specific for PNAG are purified using magnetic beads(available from Miltenyi Biotec Inc., Auburn, Calif.) and cultured invitro with PNAG. Methods to obtain PNAG and culture conditions for humanmemory B cells are described (see e.g., Kelly-Quintos et al., Ibid. andU.S. Pat. No. 7,378,276, Ibid.). Supernatants from the cultures aretested for anti-IsaA IgG antibody with an ELISA (see e.g., Lorenz etal., Ibid.) and memory B cells producing anti-IsaA antibody are selectedand expanded for adoptive immunotherapy.

The human patient at risk of MRSA infection is given recombinant memoryB cells as a therapeutic and prophylactic cell therapy which can beactivated in vivo. The recombinant memory B cells are activated in vivoby administration of S. aureus antigen to the patient when an anti-MRSAantibody response is needed. For example, approximately 10⁸-10⁹recombinant B cells may be injected as a prophylactic when the patientis healthy or has been recently infected with MRSA. The recombinantmemory B cells may be activated when needed by intracutaneous injectionof 100 μg of S. aureus antigen to the patient. For example, the memory Bcells may be activated after the patient is exposed to MRSA or at thefirst signs of infection. The production of anti-MRSA antibodies may bemonitored by sampling the patient's peripheral blood and performingELISA with MRSA antigens as the target antigens.

Example 6 Adoptive Immunotherapy of a Drug-Resistant Bacterial Infectionwith Autologous Memory B Lymphocytes Engineered to Produce Two DifferentAnti-Staphylococcus aureus Antibodies

An isolated recombinant B lymphocyte cell line that produces twodifferent secreted immunoglobulins to methicillin-resistantStaphylococcus aureus (MRSA) and produces a membrane immunoglobulin to aS. aureus antigen can be utilized for cell therapy in a mammaliansubject. The recombinant B lymphocyte cell line can be injected into themammalian subject as cell therapy to provide immunological protectionfrom infection by MRSA. The recombinant B lymphocyte cell line can beactivated in vivo or ex vivo to produce antibody to MRSA by injectingthe mammalian subject (or an in vitro cell culture) with S. aureusantigen. The timing to stimulate immunological protection from MRSAinfection in the mammalian subject can be chosen based upon the timingof an outbreak of MRSA infection in the population at large.

To protect and treat a patient with recurrent MRSA infections thepatient is given autologous recombinant B cells. The patient's memory Bcells are genetically engineered to express two antibodies recognizingtwo S. aureus antigens: poly-N-acetyl glucosamine (PNAG) andimmunodominant S. aureus antigen (IsaA). The recombinant memory B cellsare activated and expanded in vitro in culture media (e.g., RPMI 1640,Sigma-Aldrich, St. Louis, Mo.) containing: cognate antigen, PNAG, atapproximately 100 ng/mL and activating cytokines, e.g., interleukin-2(Roche, Indianapolis, Ind.), interleukin-4, interleukin-21 and ananti-CD40 antibody (R&D Systems, Minneapolis, Minn.). Afterapproximately 5 days of culture the memory B cells are harvested, washedand concentrated prior to infusion in the patient. Approximately 5×10⁸recombinant B cells are infused in the patient and the expansion andpersistence of the recombinant B cells are followed by sampling thepatient's peripheral blood. Methods to infuse and track geneticallyengineered lymphocytes are described (see e.g., Kalos et al., Sci.Transl. Med. 3, 95ra73, 2011; DOI: 10.1126/scitranslmed.3002842 which isincorporated herein by reference). For example, quantitative PCRanalysis on genomic DNA obtained from the patient's whole blood may beused to determine the copy number of the anti-PNAG Ig genes and theanti-IsaA Ig genes per microgram of genomic DNA. Approximately 100-200ng of genomic DNA is analyzed with ABI Taqman technology (available fromLife Technologies Corp., Carlsbad, Calif.). PCR primers specific for thetransfected Ig genes are validated by analysis of control genomic DNAspiked with known copy numbers of the anti-S. aureus Ig genes. Thenumber of genetically engineered B cells persisting in the peripheralblood may also be assessed using flow cytometry and fluorescentlylabeled PNAG in combination with an anti-IgG antibody. For example,phycoerythrin (PE)-conjugated PNAG and fluorescein isothiocyanate(FITC)-conjugated anti IgG are used to stain the recombinant B cells andcount them. Protocols, reagents and instrumentation for flow cytometryare available from Becton Dickinson, Franklin Lakes, N.J. In addition,the level of anti-IsaA IgG (λ) antibody in the patient's peripheralblood may be analyzed using an ELISA. The ELISA may be constructed withrecombinant purified IsaA protein and anti-IgG or anti-λ L-chainantibodies. Methods to construct and perform an ELISA are known (seee.g., Kelly-Quintos et al., Ibid.).

The recombinant memory B cells may be activated in vivo as well as invitro to produce anti-S. aureus antibodies. The memory B cells may beactivated in vivo by PNAG released from S. aureus infecting the patientor by injection of purified PNAG. Methods to purify PNAG from S. aureusare known (see e.g., Lorenz et al., Ibid.). The memory B cells areactivated in vivo by binding of PNAG to their B cell receptors (BCR) andby interaction with T cells and cytokines (see e.g., Abbas et al.,Ibid.). To enhance the activation of the recombinant memory B cells thePNAG may be administered with an immunologic adjuvant (e.g., aluminumhydroxide). Repeated activations of the memory B cells may be performedin response to recurrent MRSA infections.

The human patient at risk of MRSA infection is administered recombinantmemory B cells as a therapeutic and prophylactic cell therapy which canbe activated in vivo. The recombinant memory B cells are activated invivo by administration of PNAG antigen to the patient when an anti-MRSAantibody response is needed. For example, approximately 10⁸-10⁹recombinant B cells may be injected as a prophylactic when the patientis healthy or has been recently infected with MRSA. The recombinantmemory B cells may be activated when needed by intracutaneous injectionof 100 μg of PNAG to the patient. For example, the memory B cells may beactivated after the patient is exposed to MRSA or at the first signs ofinfection. The production of anti-MRSA antibodies may be monitored bysampling the patient's peripheral blood and performing ELISA with MRSAantigens as the target antigens. Moreover, the presence of antibodiesfor MRSA can be determined by ELISA based on conserved epitopes fromMRSA (see e.g., Ekiert et al., Ibid.).

Example 7 Construction of Cytotoxic B Cells with a Recombinant B CellReceptor

B lymphocytes produce antibodies in response to binding antigens frominfectious disease microorganisms, or cancer cells, but whenco-stimulated with antigens and selected cytokines they may also producecytotoxic molecules. For example, stimulation of B cells with thecytokine, interleukin-21 (IL-21) and antigen can result in theproduction of cytotoxic molecules (e.g., granzyme B) which may causecell death. Cytotoxic B cells that are useful for adoptive cell therapyare constructed by engineering a recombinant B cell receptor (BCR) whichrecognizes a disease associated antigen and signals to elicit expressionof cytotoxic effector functions.

A recombinant B cell receptor is constructed with a single chainantibody, membrane immunoglobulin (Ig) domains, and the cytoplasmicdomain of the IL-21 receptor. In this case, the single chain antibody, asingle chain variable fragment (SCFv), is specific for a tumorassociated antigen, prostate cancer lipid antigen (PCLA). The SCFv islinked to membrane Ig heavy chain domains including: Hinge (H), constantregion 3 (C_(H)3), transmembrane (TM) and cytoplasmic (Cyto) domainswhich participate in signaling for B cell activation, and lastly, to theIL-21 receptor cytoplasmic domain which signals to elicit cytotoxicityfunctions from the B cell. See FIG. 8A. Thus, the gene transfer andexpression of the recombinant B cell receptor in these engineered Bcells produces modified B cells that are responsive to the antigen PCLA.Upon exposure to PCLA on cancer cells, the modified B cells producecytotoxic effector molecules, such as granzyme B, and kill prostatecancer cells expressing PCLA.

The immunoglobulin (Ig) genes encoding an antibody that binds PCLA areisolated and engineered to construct a recombinant BCR gene for transferand expression in a diseased subject's own B cells. PCLA, a glycolipidantigen associated with prostate cancer is obtained from prostate cancercell lines and used as an antigen. The glycolipid antigen is used toselect a single chain antibody variable fragment (SCFv) which bindsPCLA. SCFv containing Ig variable region genes connected by a linkerpeptide have been described and can be adapted to this embodiment andmethods to select antibodies from phage display single chain variablefragment (SCFv) libraries can be utilized. A SCFv protein (and thecorresponding SCFv gene) which avidly binds PCLA on prostate cancercells is selected for construction of a recombinant BCR.

The anti-PCLA SCFv gene is attached to segments encoding domains ofmembrane IgG1 heavy (H)-chain to create a recombinant B cell receptor.The hinge segment 1008, the carboxy-terminal heavy chain constant regiondomain (C_(H)3) 1010, the transmembrane domain (TM) 1015, and thecytoplasmic domain 1020 of membrane IgG1 heavy chain are encoded at the3′ end of the SCFv 1005 gene. See FIG. 8A. Detailed methods to constructmembrane IgG H-chains can be adapted to this embodiment. The IgG1transmembrane and cytoplasmic domains are a 52 amino acid segment thatinteracts with the associated transmembrane B cell signaling proteins,Igα and Igβ, that comprise the B cell receptor [(see e.g., Abbas et al.,Cellular and Molecular Immunology, 7th Edition, pp. 159-161, 2012,Elsevier, Philadelphia, Pa.)].

The last segment of the recombinant B cell receptor contains thecytoplasmic domain of the interleukin-21 (IL-21) receptor 1025. TheIL-21 receptor can signal to elicit cytotoxic effector functions in Bcells. For example, co-stimulation of human B cells with an anti-Igantibody (i.e., stimulation of membrane IgG) and IL-21 elicitsexpression of cytotoxic effectors such as granzyme B and perforin. Thecytoplasmic domain of the IL-21 receptor protein has been identified,and the structure and signaling of the IL-21 receptor have beendescribed. See FIG. 8A for a model of the recombinant B cell receptorprotein.

DNA segments encoding the SCFv, IgG constant domains and the IL-21receptor cytoplasmic domain are amplified from DNA clones using thepolymerase chain reaction or synthesized (e.g., Custom DNA synthesis isavailable from Life Technologies Corp., Grand Island, N.Y. 14072). Thesegment encoding the anti-PCLA SCFv may be amplified from the phageclone selected above. Immunoglobulin constant region, transmembranedomain and cytoplasmic domain sequences can be synthesized by automatedDNA synthesis based on publicly available sequences, and the IL-21receptor sequence and subdomains are available. The gene encoding therecombinant B cell receptor may be assembled from the DNA segments usingthe splice overlap extension method.

The recombinant B cell receptor gene is inserted in a mammalian cellexpression vector for transfer into human B cells. An expression vectorwith cytomegalovirus (CMV) promoter elements, a selectable marker geneand poly A addition signals is described. See FIG. 8B. The plasmidvector directs expression of the recombinant B cell receptor under thecontrol of the CMV promoter and carries a selectable marker gene toallow selection of B cells expressing the vector with a drug (e.g., Neoexpression confers resistance to G418; both resistance gene, Neo, anddrug, G418, are available from InVivoGen, San Diego, Calif.). Theplasmid vector encoding the recombinant B cell receptor is transfectedinto primary human B cells (isolated from peripheral blood) using adevice, kit and protocol available from Lonza Inc., Allendale, N.J.07401 (see e.g., Human B Cell Protocol Nucleofector® Kit, which isincorporated herein by reference). Transfected, G418-resistant B cellsexpressing the recombinant B cell receptor are identified by flowcytometry using antibodies specific for the SCFv present in therecombinant B cell receptor. Alternatively PCLA antigen may be used toidentify B cells expressing the recombinant B cell receptor, forexample, by identifying and sorting cells by flow cytometry.

B cells expressing the recombinant B cell receptor are tested in vitrofor cytotoxic effector function following stimulation. Cytotoxic B cellsexpressing the anti-PCLA recombinant BCR are stimulated with antibodiesspecific for the SCFv or Ig H-chain constant region components (e.g.,anti-human IgG) of the recombinant BCR, and cell culture supernatantsare analyzed for the presence of Granzyme B by using an immunoassay. AGranzyme B ELIspot assay kit (available from Cell Sciences, Canton,Mass.) may be used to determine the number of Granzyme B producing cellsin a culture. An Immunospot Analyzer and Immunospot 3 software (CTLCellular Technology Ltd., Cleveland, Ohio) can be used to detect andcount the Granzyme B producing modified B cells.

To determine target cell killing by the recombinant cytotoxic B cells, aflow cytometry-based assay is used to determine the percentage of targetcells which are apoptotic after exposure to recombinant cytotoxic Bcells. For example, approximately 250,000 recombinant cytotoxic B cellsare added to 10,000 prostate cancer cells (e.g., PC3 cell line whichexpresses PCLA). After co-culture for approximately 3 days, target cell(i.e., PC3) apoptosis is determined by staining with annexin V andpropidium iodide; the percentage of apoptotic cells is determined byflow cytometry. A matched negative control culture with a target cellline not expressing PCLA (e.g., HeLa) is compared to the PC3 culture.Also, cultures with different ratios of effector cells (recombinantcytotoxic B cells) to target cells (PC3 cells) are analyzed. Forexample, cultures with Effector:Target ratios of 5:1, 10:1, 25:1 and50:1 are analyzed for target cell apoptosis and viability. A plot oftarget cell viability versus Effector: Target cell ratio can indicatecytotoxic effector function by the recombinant cytotoxic B cells.

Example 8 Construction of Cytotoxic B Cells with a Recombinant B CellReceptor and Coordinate Perforin Expression

Modified B cells that exhibit cytotoxicity responsive to a prostatecancer tumor antigen are engineered with a recombinant B cell receptorand an inducible gene for perforin which promote cytotoxicity for thetarget cells. Stimulation of B cells with the cytokine, interleukin-21(IL-21) and antigen can result in the production of cytotoxic molecules(e.g., granzyme B) which may cause cell death. However, expression ofperforin, an important cytotoxic effector molecule may be lacking in Bcells. Therefore, in order to provide coordinated production ofperforin, an expression cassette for perforin is placed under thecontrol of Ig heavy chain variable region (V_(H)) promoter/enhancersequences. Engineered cytotoxic B cells with coordinate expression ofgranzyme B and perforin are cytotoxic for target cells, i.e., prostatecancer cells.

A recombinant B cell receptor is constructed with a single chainantibody, membrane immunoglobulin (Ig) domains, and the cytoplasmicdomain of the IL-21 receptor. The single chain antibody, a single chainvariable fragment (SCFv), is specific for a tumor associated antigen,prostate cancer lipid antigen (PCLA). The SCFv is linked to membrane Igheavy chain domains including: Hinge (H), constant region 3 (C_(H)3),transmembrane (TM) and cytoplasmic domains, which participate insignaling B cell activation, and lastly, to the IL-21 receptorcytoplasmic domain which signals to elicit cytotoxicity functions fromthe B cell. See FIG. 8A. Gene transfer and expression of the recombinantB cell receptor expression vector (see FIG. 8B) generates B cells thatexpress the recombinant receptor, respond to PCLA, and produce cytotoxiceffector molecules such as granzyme B. A detailed description of therecombinant B cell receptor is given in Prophetic Example 1 above.

Moreover the expression vector and transfection methods to obtain Bcells expressing the recombinant B cell receptor are given (seeProphetic Example 1). Recombinant B cells respond to antigen (i.e., PCLAor tumor cells bearing PCLA on their surface) by signaling via therecombinant BCR to activate transcription at the active Ig H and L loci.For example, signaling by the recombinant BCR via interaction with Igαand Igβ may result in B cell activation and differentiation, andsignaling via the IL-21 cytoplasmic domain may lead to granzyme Bproduction. In addition, to further promote the cytotoxicity of therecombinant B cell, the human perforin gene is introduced at the activefunctional Ig H chain locus under the control of the Ig V_(H) promoterand Ig enhancer elements.

Cytotoxic B cells are engineered to express the human perforin gene fromthe active functional Ig heavy chain locus and under the control of IgV_(H) promoter sequences and an Ig enhancer element. The gene encodinghuman perforin is publicly available. The approximately 1668 nucleotidecomplementary DNA (cDNA) encoding human perforin is amplified using thepolymerase chain reaction (PCR) and oligonucleotide primers to addterminal sequences homologous to 5′ and 3′ flanking sequences of theactive, rearranged Ig gamma H-chain gene in the recombinant B cell line.See FIG. 3A. For example, the perforin cDNA is amplified with a 5′primer containing approximately 30 nucleotides homologous to upstreamsequence flanking the active V_(H) gene (see e.g., V_(H)1D₁J₂ in FIG.3A), and a 3′ primer containing approximately 30 nucleotides homologousto sequence downstream from the active constant region gene (see e.g.,C_(H)γ in FIG. 3A). The amplified perforin gene with ends homologous tothe active Ig H-chain gene on chromosome 14 is integrated by homologousrecombination to replace the γH chain gene (see FIG. 8C). Engineeringand site-specific integration of genes at the active Ig heavy chainlocus in an isolated recombinant cell line are described [(see e.g.,U.S. Pat. No. 9,175,072, Ibid.)]. Perforin expression by transfected Bcells may be determined using an Elispot assay which enumerates perforinproducing cells in vitro following stimulation of the cells. Materialsand protocols for a human perforin Elispot assay are available from CellSciences, Inc., Canton, Mass. (see Data sheet: Human Perforin ElispotKit, available online at www.cellsciences.com which is incorporatedherein by reference).

To determine target cell killing by recombinant cytotoxic B cellsexpressing perforin, a flow cytometry-based assay is used to determinethe percentage of target cells which are apoptotic after exposure torecombinant cytotoxic B cells. For example, approximately 250,000recombinant cytotoxic B cells are added to 10,000 prostate cancer cells(e.g., PC3 cell line which expresses PCLA). After co-culture forapproximately 3 days, target cell (i.e., PC3) apoptosis is determined bystaining with annexin V and propidium iodide; the percentage ofapoptotic cells is determined by flow cytometry. A matched negativecontrol culture with a target cell line not expressing PCLA (e.g., HeLa)is compared to the PC3 culture. Also, cytotoxic B cells not transfectedwith the perforin gene are compared in the cytotoxicity assay. Cultureswith different ratios of effector cells (recombinant cytotoxic B cells)to target cells (PC3 cells) are analyzed. For example, cultures withEffector:Target ratios of 5:1, 10:1, 25:1 and 50:1 are analyzed fortarget cell apoptosis and viability. A plot of target cell viabilityversus Effector:Target cell ratio can indicate cytotoxic effectorfunction by the recombinant cytotoxic B cells.

Example 9 Engineered B Lymphocytes Express a Recombinant B-Cell ReceptorSpecific for Prostate Cancer Lipid Antigen and a Secreted AntibodySpecific for Prostate Specific Stem Cell Antigen

An isolated recombinant B lymphocyte cell line that expresses arecombinant B cell receptor (BCR) specific for prostate cancer lipidantigen (PCLA) and secretes an antibody recognizing prostate stem cellantigen (PSCA) is constructed for therapy of prostate cancer. Therecombinant B lymphocyte cell line is infused in a subject to provide Bcells that are not only cytotoxic for tumor cells but also produce atherapeutic antibody that recognizes prostate cancer cells. RecombinantB lymphocyte cells bind PCLA on prostate cancer cells via an engineeredrecombinant BCR and are activated to produce cytotoxic effectors (e.g.,Granzyme B) and to secrete anti-PSCA antibody.

The recombinant B cells provide cellular and humoral immunity targetedto prostate cancer cells. The recombinant B lymphocyte cell line canalso be stimulated in vivo or ex vivo by injecting the mammalian subject(or an in vitro cell culture) with exogenous PCLA to elicit cytotoxiceffectors and to produce secreted anti-PSCA antibody. Determination oftiming to stimulate immunological reactivity to prostate cancer cells inthe mammalian subject can be chosen based upon the detection of prostatecancer cells in the mammalian subject.

Polyclonal memory B cells expressing B cell receptors (BCR) of membraneIgG are isolated from a prostate cancer patient. Polyclonal memory Bcells are isolated from the patient's peripheral blood: 1) by isolatingperipheral blood mononuclear cells using Ficoll Hypaque densitygradients (available from Sigma Aldrich. St. Louis. Mo.); 2) by negativeselection of total B cells using magnetic beads (available from StemCell Technology Vancouver, BC), and 3) by labeling the cells withfluorescent monoclonal antibodies that recognize membrane IgG and CD27,a memory B cell marker, and performing fluorescence-activated cellsorting. Memory B cells expressing membrane IgG are cultured in vitroand genetically engineered to express a recombinant B cell receptor anda secreted anti-PCSA antibody.

Memory B cells are genetically engineered to express a recombinant BCRspecific for PCLA and a secreted IgG antibody specific for prostate stemcell antigen (PSCA). An isolated recombinant cell line is constructedthat includes a recombinant BCR. The recombinant BCR binds PCLA onprostate cancer cells and signals intracellularly to the B cell toelicit expression and release of cytotoxic effector molecules such asGranzyme B as described above in Prophetic Example 6. The recombinantcell line may be selected using drug selection, e.g., G418, and flowcytometry to identify clones expressing the recombinant BCR (see e.g.,Prophetic Example 6 above).

Immunoglobulin genes encoding an anti-PSCA antibody (i.e., Ig heavychain and light chain genes) are integrated at the active, rearranged Igheavy chain locus on chromosome 14 (see FIG. 3A), and the active,rearranged kappa light chain locus on chromosome 2, respectively.Methods and materials to obtain anti-PSCA antibodies are available. Forexample, to target integration of anti-PSCA antibody genes into thefunctional γ1-H chain locus, targeting sequences from the intron betweenthe J_(H) exons and the γ constant region gene (C_(H)γ; see FIG. 3A) areplaced 5′ of the anti-PSCA γ1-H chain gene and targeting sequencesselected downstream (3′) from the γ1-H chain cytoplasmic exon are placed3′ of the γ1-H chain gene (see FIG. 3A). Analogous targeting sequences(i.e., from the Jk-Ck intron and 3′ of the Ck gene) are used fortargeting the anti-PSCA kappa L chain gene into the functional Ck gene.The targeting vectors for anti-PSCA H and L chain include selectablemarker genes, e.g., hygromycin resistance and histidinol dehydrogenase,respectively. Media containing hygromycin and histidinol are used toselect for engineered memory B cells expressing the targeting vectorsencoding a secreted IgG anti-PSCA antibody.

Following transfection and selection of the recombinant memory B cells,those cells producing secreted IgG antibodies specific for PSCA areidentified using standard immunoassays to assess B cell supernatants.The engineered memory B cells are cultured in vitro and stimulated withPCLA to activate the cell cytotoxicity and to stimulate secretion ofanti-PSCA IgG antibodies. Laboratory methods to purify PCLA, the lipidantigen, can be adapted for this embodiment. Also, isolated recombinantcell lines expressing the recombinant B cell receptor and anti-PSCAantibodies are tested in a cytotoxicity assay with target cellsexpressing PCLA and PSCA. For example, PC3, a prostate tumor cell linebearing PCLA can be transduced with a vector encoding PSCA and tested invitro in cytotoxicity assays with engineered cytotoxic B cell lines.Methods and materials for transducing PC3 cells are described, anddetails of the cytotoxicity assay are described above. See PropheticExample 6.

Example 10 Construction of Cytotoxic B Cells for Prostate Cancer byTransfection of B Cells with Transcription Factors

Cytotoxic B cells are produced by engineering memory B cells expressingB cell receptors (BCR) specific for prostate cancer lipid antigen(PCLA). The B cells are transfected with a viral vector encodingtranscription factors which control the expression of cytotoxic effectormolecules including granzyme B and perforin. Engineered cytotoxic Bcells that recognize and kill prostate cancer cells can be used foradoptive cell therapy in prostate cancer patients.

Memory B cells expressing B cell receptors recognizing PCLA areconstructed with immunoglobulin (Ig) genes encoding an anti-PCLAantibody. The engineered Ig genes are inserted at the activelytranscribed heavy (H) and light (L) chain loci on human chromosomes 14and 2. Polyclonal memory B cells expressing B cell receptors (BCR) ofmembrane IgG are isolated from a prostate cancer patient. Polyclonalmemory B cells are isolated from the patient's peripheral blood: 1) byisolating peripheral blood mononuclear cells using Ficoll Hypaquedensity gradients (available from Sigma Aldrich. St. Louis. Mo.); 2) bynegative selection of total B cells using magnetic beads (available fromStem Cell Technology Vancouver, BC), and 3) by labeling the cells withfluorescent monoclonal antibodies that recognize membrane IgG and CD27,a memory B cell marker, and performing fluorescence-activated cellsorting. Memory B cells expressing membrane IgG are cultured in vitroand genetically engineered to express a B cell receptor specific forPCLA.

Immunoglobulin genes encoding an anti-PCLA antibody (i.e., Ig heavychain and light chain genes) are integrated at the active, rearranged Igheavy chain locus on chromosome 14 (see FIG. 3A), and the active,rearranged kappa light chain locus on chromosome 2, respectively.Anti-PCLA antibodies are obtained, and the corresponding Ig genes areisolated, engineered and site-specifically integrated at the active Igheavy chain and light chain loci in an isolated recombinant cell line.For example, to target integration of anti-PCLA membrane Ig H-chain geneinto the functional, rearranged γ-H chain locus: Targeting sequencesupstream (5′) from the variable region exon (VH1D1J2; see FIG. 3A) anddownstream (3′) from the gamma-H chain constant region exon (CHγ; seeFIG. 3A) are placed 5′ and 3′ respectively, of the engineered, membraneγ-H chain gene (see FIG. 3B). Analogous targeting sequences (i.e., from5′ of VkJk exon and 3′ of the Ck gene) are used for targeting theanti-PCLA kappa L chain gene into the functional Ck gene on Chromosome2.

Engineered B cells expressing membrane IgG specific for PCLA aretransduced with a viral vector encoding three transcription factorsessential for cytotoxic B cell function. Three transcription factorsregulate the expression of cytotoxic effector molecules such as granzymeB. Transcription factors: T-bet, Runx3 and Eomes are essential for theexpression of granzyme B and perforin in the context of cytotoxic T celldifferentiation. A tricistronic vector encoding T-bet, Runx3 and Eomesis constructed using a Sendai virus vector which transduces human Bcells. See FIG. 8D. Transfection and expression of the vector in theengineered B cells can be monitored by immunostaining withantibody-enzyme conjugates specific for T-bet, Runx3 and Eomes (e.g.,anti-Eomes, anti-T-bet and anti-Runx3 antibodies are available fromAbcam, Cambridge, Mass.), and expression of Granzyme B and perforin canbe detected by reverse transcriptase-polymerase chain reaction (RT-PCR)of RNA from the transfected cells.

Engineered cytotoxic B cells expressing membrane IgG specific for PCLAare tested for cytotoxic effector function versus prostate cancer celllines. For example, PC3, a prostate tumor cell line bearing PCLA can beused as a target cell in a flow cytometric assay that detects apoptoticcells following exposure to the engineered cytotoxic B cells (seeProphetic Example 2 above).

Engineered cytotoxic B cells expressing membrane IgG recognizing PCLAcan also be tested in vivo in a xenogeneic mouse model of human prostatecancer. For example, PC3, human prostate tumor cells are implantedsubcutaneously in immunodeficient mice (e.g., NSG mice available fromJackson Labs, Bar Harbor, Me.) and the mice are treated with theengineered cytotoxic B cells. The mice are evaluated with respect totumor size, body weight and survival. Control tumor cell lines caninclude tumors not expressing PCLA. Moreover, the survival or expansionof cytotoxic B cells in the mice following infusion or injection incontrol mice or PC3 tumor-bearing mice is evaluated and recorded.

Example 11 Modified Memory B Lymphocytes to Treat Prostate Cancer

Memory B lymphocytes are isolated from a prostate cancer patient andengineered to express an anti-PCLA (prostate cancer lipid antigen)membrane Ab (antibody) and a chemokine receptor, CXCR3. A gene encodinga single chain anti-PCLA Ab is integrated at the active, rearrangedkappa light (L)-chain locus on chromosome 2, and disrupts endogenous Igkappa L-chain expression. The CXCR3 gene is integrated at the endogenousactive, rearranged immunoglobulin (Ig) heavy (H)-chain locus anddisrupts endogenous Ig H-chain expression. Engineered B cells that hometo prostate cancer tumors by virtue of CXCR3-mediated chemotaxis (seee.g., Sackstein et al., Laboratory Investigation 97: 669-697, 2017 whichis incorporated herein by reference) are used for adoptive cell therapyof prostate cancer. Tumor-localized, engineered B cells providetherapeutic anti-PCLA antibodies to tumor cells at elevated localconcentrations.

Memory B cells are engineered to express a single chain membrane Ab forPCLA (prostate cancer lipid antigen) under the control of the endogenousIg kappa promoter and/or enhancer sequences by insertion in theendogenous rearranged, active kappa light chain gene locus using CRISPRtechnology (Clustered Regularly Interspaced Short Palindromic Repeats).The immunoglobulin (Ig) genes encoding an antibody that binds PCLA areisolated and engineered to construct a recombinant Ig gene for transferand expression in a diseased subject's own B cells. PCLA, a glycolipidantigen associated with prostate cancer is obtained from prostate cancercell lines and used as an antigen. The glycolipid antigen is used toselect a single chain antibody variable fragment (SCFv) which bindsPCLA. SCFv containing Ig variable region genes connected by a linkerpeptide have been described and can be adapted to this embodiment.

Methods to select antibodies from phage display single chain variablefragment (SCFv) libraries can be utilized. A SCFv protein (and thecorresponding SCFv gene) which avidly binds PCLA on prostate cancercells is selected for construction of a recombinant single chain Ab. Theanti-PCLA SCFv is connected to a γ1-H chain constant region gene thatencodes the transmembrane and cytoplasmic domains of the γ1-H chain. Theanti-PCLA single chain Ab gene is incorporated in an AAV vector(Adeno-Associated Virus vector) and is flanked by homology arms forintegration at the kappa L-chain locus on chromosome 2 (see e.g., Eyquemet al., Nature 543, 113-117, 2017 which is incorporated herein byreference). Homology arms that target the active endogenous Igkappa-chain gene flank the anti-PCLA Ab gene to direct integration intothe kappa L-chain locus and disrupt endogenous kappa L-chain expression.See FIG. 9A for more details.

Synthetic RNAs encoding a guide RNA targeting the integration site and amessenger RNA encoding Cas9 endonuclease are introduced byelectroporation into memory B cells. Approximately 2 hours later thecells are transduced with the AAV-PCLA Ab vector. (Synthetic guide RNAsand Cas9 mRNA are available from Trilink Biotechnologies, San Diego,Calif., and AAV vectors are available from Cell Biolabs, Inc., SanDiego, Calif.).

Memory B cells are engineered to also express a gene for CXCR3 at theactive Ig H-chain locus on chromosome 14. Simultaneous integration ofthe anti-PCLA Ab and CXCR3 genes using CRISPR technology can beperformed to expedite and optimize engineering of memory B cells (seee.g., Le Cong et al., Science 339: 819-823, 2013 which is incorporatedherein by reference). A gene encoding CXCR3 is available from GenScript,Piscataway, N.J. Site-specific integration of the CXCR3 gene at theactive H-chain locus is accomplished using CRISPR technology (see e.g.,Eyquem et al., Nature 543: 113-117, 2017 which is incorporated herein byreference). An adenovirus-associated viral (AAV) vector is designed toencode the human CXCR3 gene flanked by homology arms targeting the IgH-chain CH1 exon on Chromosome 14, in order to disrupt the active IgH-chain gene and insert a functional CXCR3 gene. See FIG. 9B for moredetails.

Synthetic RNAs encoding guide RNAs targeting the integration site and amessenger RNA encoding Cas9 endonuclease are introduced byelectroporation into memory B cells isolated from the prostate cancerpatient. Approximately 2 hours later the cells are transduced with theAAV-CXCR3 vector. (Synthetic guide RNAs and Cas9 mRNA are available fromTrilink Biotechnologies, San Diego, Calif., and AAV vectors areavailable from Cell Biolabs, Inc., San Diego, Calif.). Geneticallyengineered B cells are grown in vitro using culture media containingcytokines such as IL-21 and co-stimulators such as oligodeoxynucleotide,CpG and anti-CD40 Ab (see e.g., Kwakkenbos et al., Nature Medicine 16:123-128, 2010 which is incorporated herein by reference).

B cells expressing CXCR3 from the disrupted Ig H-chain locus areidentified and isolated using flow cytometry. B cells that stain withfluorescent anti-CXCR3 Ab and are not stained by fluorescent anti-IgMantibody are selected by cell sorting and cultured in vitro. Fluorescentanti-CXCR3 and anti-human IgM Abs are available from ABCAM, Cambridge,Mass. B cells staining positive with CXCR3 Ab are tested in vitro forexpression of CXCR3 mRNA using RT-PCR; B cells negative for CXCR3 and/orpositive for IgM are tested as negative controls. Each B cell sample istested plus and minus stimulation with B cell activators (e.g., IL-21,anti-CD40 and CpG).

To evaluate biological function of the CXCR3 gene in engineered B cells,an in vitro chemotaxis assay is performed with CXCL10, a ligand forCXCR3. Engineered B cells are introduced onto a permeable membrane in atranswell plate. The chamber below the membrane is filled with culturemedium containing CXCL10 and following incubation for several hours themigration of engineered B cells to the lower chamber is scored bystaining the B cells in situ. Control plates omit CXCL10, or usenon-transfected B cells or IgM-positive B cells. Methods and materialsto perform the chemotaxis assay are described (see e.g., Conley-LaCombet al., Ibid. and Xia et al., Oncotarget 7: 60461-474, 2016 which areincorporated herein by reference).

The recombinant memory B cells are activated and expanded in vitro inculture media (e.g., RPMI 1640, Sigma-Aldrich, St. Louis, Mo.)containing: cognate antigen, PCLA, at approximately 100 ng/mL andactivating cytokines, e.g., interleukin-2 (Roche, Indianapolis, Ind.),interleukin-4, interleukin-21 and an anti-CD40 antibody (R&D Systems,Minneapolis, Minn.).

After approximately 5 days of culture the memory B cells are harvested,washed and concentrated prior to infusion in the patient. Approximately5×10⁸ recombinant B cells are infused and the expansion and persistenceof the recombinant B cells are followed by sampling the patient'speripheral blood. Methods to infuse and track genetically engineeredlymphocytes are described (see e.g., Kalos et al., Sci. Transl. Med. 3,95ra73, 2011; DOI: 10.1126/scitranslmed.3002842 which is incorporatedherein by reference).

Example 12 Construction of Engineered Memory B Lymphocytes to ModulateAutoimmunity

Memory B lymphocytes obtained from a patient with multiple sclerosis(MS) are engineered to express a membrane antibody (Ab) that recognizesmyelin oligodendrocyte glycoprotein (MOG), and to produce ananti-inflammatory cytokine, interleukin 10 (IL-10). The engineered Bcells bind MOG and respond by producing IL-10. A gene for an anti-MOGmembrane Ab is expressed under the control of a constitutive promoter,and a gene encoding IL-10 is integrated at the endogenous Ig heavy(H)-chain locus of memory B cells and expressed under the control of theendogenous rearranged immunoglobulin variable heavy (VH) promoter and/orenhancer elements.

A gene encoding IL-10 is inserted at the endogenous active Ig H-chainlocus on chromosome 14 using CRISPR technology. For example acomplementary DNA (cDNA) encoding human IL-10 (available from HarvardMedical School, Boston, Mass.) is incorporated in an adenovirusassociated virus (AAV) vector (e.g., AAV vectors are available from CellBiolabs, Inc., San Diego, Calif.). The human IL-10 cDNA is flanked by asplice acceptor (SA) sequence, a poly A addition site (pA) and homologyarms (HA) to target recombination at the Ig H-chain locus on Chromosome14. See FIG. 12A for more details.

A guide RNA targeting the CHμ1 exon of the μ H-chain gene results in adouble-stranded DNA break that disrupts μ H-chain expression andpromotes insertion of the IL-10 gene. See FIG. 12A for more details.

Methods to construct guide RNAs for targeted insertion are described(see e.g., Eyquem et al., Ibid. and Zheng et al., BioTechniques57:115-124, 2014 which is incorporated herein by reference), andsynthetic guide RNAs and Cas9 mRNA are available from TrilinkBiotechnologies, San Diego, Calif. Memory B cells expressing membraneIgM are obtained and purified from the peripheral blood of a MS patient.Memory B cells are obtained by cell sorting using anti-IgM and anti-CD27antibodies. For example, see Tangye et al., J. Immunology 179, 13-19,2007 which is incorporated herein by reference). Methods and materialsfor electroporation and viral transduction of lymphocytes are described(see e.g., Eyquem et al., Ibid.). Memory B cells that lack membrane IgMon their surface are tested for their production of IL-10. Membrane IgMnegative, IL-10 positive B cells are expanded and activated prior tolentivirus transduction.

A lentiviral vector is constructed to direct expression of an anti-MOGmembrane Ab on memory B cells. For example, a single chain, membrane Abgene may be constructed with a gene encoding a human single chainvariable fragment (SCFv) specific for MOG (obtained from a SCFv phagelibrary). The SCFv gene is joined to a human Igγ1 constant region genewhich includes: the hinge region, C_(H)1-CO, the transmembrane domain(TM) and the cytoplasmic tail (Cyto) (see e.g., Xu et al., Cell Research24, 651-654, 2014 which is incorporated herein by reference). See FIG.12B.

The anti-MOG membrane Ig gene is preceded by a constitutive promoter,(e.g., the human cytomegalovirus promoter (CMV) and cloned in alentiviral vector suitable for transduction of B lymphocytes (see e.g.,Bovia et al., Blood 101, 1727-1733, 2003 which is incorporated herein byreference). Methods and reagents to activate and transduce human Blymphocytes are described (Bovia et al., Ibid.). B lymphocytesexpressing membrane IgG are detected and purified using anti-human IgGantibodies (available from ABCAM Inc., Cambridge, Mass.) and flowcytometry.

Engineered B cells expressing a membrane IgG specific for MOG are testedin vitro to assess functionality. For example, the engineered B cellsare grown with media containing MOG glycoprotein to activate theirmembrane IgG receptors and media samples are collected and assayed forIL-10. Control cultures containing human serum albumin, or no additionalprotein are also tested. Positive control cultures containing anti-humanIgG are also tested for IL-10 concentration. Standard enzyme-linkedimmunosorbent assays for IL-10 and recombinant human MOG protein areavailable from R&D Systems, Inc., Minneapolis, Minn. Initial in vitrocultures to demonstrate the functionality of the engineered B cellsestablish the dose-response curve to MOG protein and the kinetics ofIL-10 production. Such dosages can be adapted for animal studies andclinical trials in human subjects.

Example 13 Construction of Cytotoxic B Cells to Treat HIV

Genes encoding a recombinant B cell receptor (BCR) and interleukin 21(IL-21) are inserted at the immunoglobulin (Ig) heavy (H)-chain locus inmemory B cells to disrupt the endogenous, active gamma (γ) H-chain geneand to engage the Ig variable region (VH) promoter and H-chain enhancerelements. Genomic targeting with CRISPR/Cas9 technology, includingspecific guide RNAs restricts DNA DS breaks and gene insertion to therearranged γ H-chain gene expressed by each IgG-positive memory B cell.

A bicistronic gene composed of a recombinant BCR gene and a gene forIL-21 are inserted in the rearranged, active γ H-chain gene onchromosome 14 in IgG-positive memory B cells. Polyclonal memory B cellsexpressing membrane IgG are isolated from the peripheral blood of apatient using magnetic bead separations and flow cytometry to isolateIgG-positive, CD27-positive, B cells (see e.g., Ettinger et al., Ibid.and Brown et al., Ibid.). Targeted disruption and integration atmultiple different active Ig γ H-chain genes is accomplished using aCRISPR/Cas9 system with two guide RNAs and a template gene forhomologous recombination. Methods and materials to delete a DNA segmentand replace it with donor gene(s) of interest are described (see e.g.,Zheng et al., BioTechniques 57, 115-124, 2014 which is incorporatedherein by reference). For example, guide RNAs (gRNAs) targeting twoconserved sites in each rearranged γ H-chain gene are designed totarget: (1) a site in VDJ-Cγ intron which is downstream (3′) of the Igmu enhancer (μEnh) but upstream (5′) of the class switch recombinationsite (CSRS); and (2) a site near the first exon (CHγ1) of the γ constantregion gene. See FIG. 13 for more details.

The design of guide RNAs and selection of target sites to give optimalspecificity and efficient deletion of genomic segments is described (seee.g., Zheng et al., Ibid.).

An AAV vector containing a bicistronic DNA construct encoding arecombinant BCR and IL-21 is constructed with homology arms that flankthe deletion in the γ 1-H chain gene. See FIG. 13 for more details.

A P2a peptide is encoded 5′ of the BCR and the IL-21 genes. Bicistronicconstructs with P2a self-cleaving peptides are described (see e.g., Kimet al., PLoS ONE 6(4): e18556, 2011; doi:10.1371/journal.pone.0018556which is incorporated herein by reference). The recombinant BCR may beconstructed from a broadly neutralizing Ab for HIV (see e.g., Balazs etal., Nature 481, 81-84, 2012 which is incorporated herein by reference)and expressed as a single chain variable fragment (SCFV).

The engineered memory B cells are incubated with HIV envelope proteinand media samples are collected at 1, 2, 4, and 8 hours and the amountof IL-21 is determined using an immunoassay (e.g., enzyme-linkedimmunosorbent assay, (ELISA)). An IL-21 ELISA kit is available fromRayBiotech, Inc., Norcross, Ga. Negative control cultures without HIVenv protein or with a control protein, (e.g., human serum albumin) arecompared to the HIV env-stimulated cultures. Positive control culturesare run using anti-IgG antibodies to stimulate the engineered B cells.

Engineered memory B cells are tested for cytotoxic effector function invitro using engineered mammalian cells expressing HIV env protein. Amammalian cell line, e.g., HEK 293 is transfected with an expressionvector encoding HIV-1 gp41 (gp41 cDNA is available from Bioclone Inc.,San Diego, Calif.) and a cell line (gp41/HEK) expressing gp41 on thecell surface is selected using an anti-gp41 monoclonal antibody, Mab.Co-culture of engineered memory B cells with gp41/HEK target cells isfollowed by monitoring cell viability, e.g., Trypan Blue staining,assessing apoptosis and measuring release lactic dehydrogenase.Moreover, the release of Granzyme B into the media can be measured usingan ELISA. The cellular cytotoxicity measured against gp41/HEK cellsversus control HEK 293 cells is obtained at various ratios of cytotoxicB cell effectors to target cells (e.g., 1:1, 2:1, 4:1, 10:1, 20:1,40:1).

Each recited range includes all combinations and sub-combinations ofranges, as well as specific numerals contained therein.

All publications and patent applications cited in this specification areherein incorporated by reference to the extent not inconsistent with thedescription herein and for all purposes as if each individualpublication or patent application were specifically and individuallyindicated to be incorporated by reference for all purposes.

Those having ordinary skill in the art will recognize that the state ofthe art has progressed to the point where there is little distinctionleft between hardware and software implementations of aspects ofsystems; the use of hardware or software is generally (but not always,in that in certain contexts the choice between hardware and software canbecome significant) a design choice representing cost vs. efficiencytradeoffs. Those having ordinary skill in the art will recognize thatthere are various vehicles by which processes and/or systems and/orother technologies disclosed herein can be effected (e.g., hardware,software, and/or firmware), and that the preferred vehicle will varywith the context in which the processes and/or systems and/or othertechnologies are deployed. For example, if a surgeon determines thatspeed and accuracy are paramount, the surgeon may opt for a mainlyhardware and/or firmware vehicle; alternatively, if flexibility isparamount, the implementer may opt for a mainly software implementation;or, yet again alternatively, the implementer may opt for somecombination of hardware, software, and/or firmware. Hence, there areseveral possible vehicles by which the processes and/or devices and/orother technologies disclosed herein may be effected, none of which isinherently superior to the other in that any vehicle to be utilized is achoice dependent upon the context in which the vehicle will be deployedand the specific concerns (e.g., speed, flexibility, or predictability)of the implementer, any of which may vary. Those having ordinary skillin the art will recognize that optical aspects of implementations willtypically employ optically-oriented hardware, software, and or firmware.

In a general sense the various aspects disclosed herein which can beimplemented, individually and/or collectively, by a wide range ofhardware, software, firmware, or any combination thereof can be viewedas being composed of various types of “electrical circuitry.”Consequently, as used herein “electrical circuitry” includes, but is notlimited to, electrical circuitry having at least one discrete electricalcircuit, electrical circuitry having at least one integrated circuit,electrical circuitry having at least one application specific integratedcircuit, electrical circuitry forming a general purpose computing deviceconfigured by a computer program (e.g., a general purpose computerconfigured by a computer program which at least partially carries outprocesses and/or devices disclosed herein, or a microdigital processingunit configured by a computer program which at least partially carriesout processes and/or devices disclosed herein), electrical circuitryforming a memory device (e.g., forms of random access memory), and/orelectrical circuitry forming a communications device (e.g., a modem,communications switch, or optical-electrical equipment). The subjectmatter disclosed herein may be implemented in an analog or digitalfashion or some combination thereof.

At least a portion of the devices and/or processes described herein canbe integrated into a data processing system. A data processing systemgenerally includes one or more of a system unit housing, a video displaydevice, memory such as volatile or non-volatile memory, processors suchas microprocessors or digital signal processors, computational entitiessuch as operating systems, drivers, graphical user interfaces, andapplications programs, one or more interaction devices (e.g., a touchpad, a touch screen, an antenna, etc.), and/or control systems includingfeedback loops and control motors (e.g., feedback for sensing positionand/or velocity; control motors for moving and/or adjusting componentsand/or quantities). A data processing system may be implementedutilizing suitable commercially available components, such as thosetypically found in data computing/communication and/or networkcomputing/communication systems.

The foregoing detailed description has set forth various embodiments ofthe devices and/or processes via the use of block diagrams, flowcharts,and/or examples. Insofar as such block diagrams, flowcharts, and/orexamples contain one or more functions and/or operations, it will beunderstood by those within the art that each function and/or operationwithin such block diagrams, flowcharts, or examples can be implemented,individually and/or collectively, by a wide range of hardware, software,firmware, or virtually any combination thereof. In one embodiment,several portions of the subject matter described herein may beimplemented via Application Specific Integrated Circuits (ASICs), FieldProgrammable Gate Arrays (FPGAs), digital signal processors (DSPs), orother integrated formats. However, some aspects of the embodimentsdisclosed herein, in whole or in part, can be equivalently implementedin integrated circuits, as one or more computer programs running on oneor more computers (e.g., as one or more programs running on one or morecomputer systems), as one or more programs running on one or moreprocessors (e.g., as one or more programs running on one or moremicroprocessors), as firmware, or as virtually any combination thereof,and that designing the circuitry and/or writing the code for thesoftware and or firmware would be well within the skill of one of skillin the art in light of this disclosure. In addition, the mechanisms ofthe subject matter described herein are capable of being distributed asa program product in a variety of forms, and that an illustrativeembodiment of the subject matter described herein applies regardless ofthe particular type of signal bearing medium used to actually carry outthe distribution. Examples of a signal bearing medium include, but arenot limited to, the following: a recordable type medium such as a floppydisk, a hard disk drive, a Compact Disc (CD), a Digital Video Disk(DVD), a digital tape, a computer memory, etc.; and a transmission typemedium such as a digital and/or an analog communication medium (e.g., afiber optic cable, a waveguide, a wired communications link, a wirelesscommunication link (e.g., transmitter, receiver, transmission logic,reception logic, etc.), etc.).

The herein described components (e.g., steps), devices, and objects andthe description accompanying them are used as examples for the sake ofconceptual clarity and that various configuration modifications usingthe disclosure provided herein are within the skill of those in the art.Consequently, as used herein, the specific examples set forth and theaccompanying description are intended to be representative of their moregeneral classes. In general, use of any specific example herein is alsointended to be representative of its class, and the non-inclusion ofsuch specific components (e.g., steps), devices, and objects hereinshould not be taken as indicating that limitation is desired.

With respect to the use of substantially any plural or singular termsherein, the reader can translate from the plural to the singular or fromthe singular to the plural as is appropriate to the context orapplication. The various singular/plural permutations are not expresslyset forth herein for sake of clarity.

The herein described subject matter sometimes illustrates differentcomponents contained within, or connected with, different othercomponents. It is to be understood that such depicted architectures aremerely examples, and that in fact many other architectures can beimplemented which achieve the same functionality. In a conceptual sense,any arrangement of components to achieve the same functionality iseffectively “associated” such that the desired functionality isachieved. Hence, any two components herein combined to achieve aparticular functionality can be seen as “associated with” each othersuch that the desired functionality is achieved, irrespective ofarchitectures or intermedial components. Likewise, any two components soassociated can also be viewed as being “operably connected,” or“operably coupled,” to each other to achieve the desired functionality,and any two components capable of being so associated can also be viewedas being “operably couplable,” to each other to achieve the desiredfunctionality. Specific examples of operably couplable include but arenot limited to physically mateable or physically interacting componentsor wirelessly interactable or wirelessly interacting components orlogically interacting or logically interactable components.

While particular aspects of the present subject matter described hereinhave been shown and described, changes and modifications may be madewithout departing from the subject matter described herein and itsbroader aspects and, therefore, the appended claims are to encompasswithin their scope all such changes and modifications as are within thetrue spirit and scope of the subject matter described herein.Furthermore, it is to be understood that the invention is defined by theappended claims. It will be understood that, in general, terms usedherein, and especially in the appended claims (e.g., bodies of theappended claims) are generally intended as “open” terms (e.g., the term“including” should be interpreted as “including but not limited to,” theterm “having” should be interpreted as “having at least,” the term“includes” should be interpreted as “includes but is not limited to,”etc.). It will be further understood that if a specific number of anintroduced claim recitation is intended, such an intent will beexplicitly recited in the claim, and in the absence of such recitationno such intent is present. For example, as an aid to understanding, thefollowing appended claims may contain usage of the introductory phrases“at least one” and “one or more” to introduce claim recitations.However, the use of such phrases should not be construed to imply thatthe introduction of a claim recitation by the indefinite articles “a” or“an” limits any particular claim containing such introduced claimrecitation to inventions containing only one such recitation, even whenthe same claim includes the introductory phrases “one or more” or “atleast one” and indefinite articles such as “a” or “an”; the same holdstrue for the use of definite articles used to introduce claimrecitations. In addition, even if a specific number of an introducedclaim recitation is explicitly recited, such recitation should typicallybe interpreted to mean at least the recited number (e.g., the barerecitation of “two recitations,” without other modifiers, typicallymeans at least two recitations, or two or more recitations).Furthermore, in those instances where a convention analogous to “atleast one of A, B, and C, etc.” is used, in general such a constructionis intended in the sense one having skill in the art would understandthe convention (e.g., “a system having at least one of A, B, and C”would include but not be limited to systems that have A alone, B alone,C alone, A and B together, A and C together, B and C together, or A, B,and C together, etc.). In those instances where a convention analogousto “at least one of A, B, or C, etc.” is used, in general such aconstruction is intended in the sense one having skill in the art wouldunderstand the convention (e.g., “a system having at least one of A, B,or C” would include but not be limited to systems that have A alone, Balone, C alone, A and B together, A and C together, B and C together, orA, B, and C together, etc.). Virtually any disjunctive word and/orphrase presenting two or more alternative terms, whether in thedescription, claims, or drawings, should be understood to contemplatethe possibilities of including one of the terms, either of the terms, orboth terms. For example, the phrase “A or B” will be understood toinclude the possibilities of “A” or “B” or “A and B.”

While various aspects and embodiments have been disclosed herein, otheraspects and embodiments will be apparent to those skilled in the art.The various aspects and embodiments disclosed herein are for purposes ofillustration and are not intended to be limiting, with the true scopeand spirit being indicated by the following claims.

What is claimed is:
 1. An isolated modified B cell capable ofexpressing: at least one exogenously incorporated membraneimmunoglobulin or a recombinant B cell receptor that is capable ofbinding to a first antigen; at least one exogenously incorporatedreassigned biological agent; and at least one endogenous secretedimmunoglobulin reactive to a second antigen.
 2. The isolated modified Bcell of claim 1, wherein the at least one exogenously incorporatedmembrane immunoglobulin includes one or more exogenously incorporatedmembrane immunoglobulin polypeptides.
 3. The isolated modified B cell ofclaim 1, wherein the at least one exogenously incorporated membraneimmunoglobulin includes at least one exogenously incorporated nucleicacid encoding the at least one exogenous membrane immunoglobulin.
 4. Theisolated modified B cell of claim 3, wherein the exogenouslyincorporated nucleic acid encoding the at least one membraneimmunoglobulin is integrated in one or more chromosomal loci in theisolated B lymphocyte cell line.
 5. The isolated modified B cell ofclaim 4, wherein the chromosomal loci include at least one of the Ig Hchain or Ig L chain chromosomal loci.
 6. The isolated modified B cell ofclaim 5, wherein the Ig H chain or Ig L chain chromosomal loci arelocated on the non-expressed Ig allele.
 7. The isolated modified B cellof claim 6, wherein at least one of the Ig H chain or Ig L chainchromosomal loci is under expression control of an endogenous promoteror enhancer elements.
 8. The isolated modified B cell of claim 6,wherein the Ig L chain locus includes at least one of the kappa orlambda light (L)-chain locus.
 9. The isolated modified B cell of claim8, wherein the integration at the kappa or lambda light (L)-chain locusdisrupts endogenous B cell kappa or lambda light (L)-chain expression.10. The isolated modified B cell of claim 4, wherein the chromosomalloci include one or more non-Ig L or non-Ig H chromosomal loci in theisolated B lymphocyte cell line.
 11. (canceled)
 12. (canceled) 13.(canceled)
 14. (canceled)
 15. The isolated modified B cell of claim 4,wherein the at least one exogenously incorporated membraneimmunoglobulin nucleic acid includes an exogenous bicistronic expressionconstruct.
 16. (canceled)
 17. (canceled)
 18. The isolated modified Bcell of claim 1, wherein the at least one exogenously incorporatedmembrane immunoglobulin includes a nucleic acid encoding one singlechain Fv immunoglobulin.
 19. The isolated modified B cell of claim 1,wherein the isolated modified B cell includes at least one of a naïve Blymphocyte, immature B lymphocyte, transitional B lymphocyte, mature Blymphocyte, B1 B lymphocyte, marginal zone B lymphocyte, follicular Blymphocyte, memory B lymphocyte, plasmablast, or plasma cell.
 20. Theisolated modified B cell of claim 1, wherein the isolated modified Bcell is part of a polyclonal population of B lymphocytes.
 21. Theisolated modified B cell of claim 1, wherein the isolated modified Bcell is part of a monoclonal population of B lymphocytes.
 22. Theisolated modified B cell of claim 1, wherein the isolated modified Bcell is part of a modified B lymphocyte cell line.
 23. The isolatedmodified B cell of claim 1, wherein the membrane immunoglobulin includesat least one of a membrane anchor, a cytoplasmic domain, a hinge regionor an extracellular ligand-binding domain.
 24. The isolated modified Bcell of claim 1, wherein the isolated modified B cell bears at least onechimeric B cell receptor capable of transducing signals for inducingexpression of cytotoxic effector molecules.
 25. The isolated modified Bcell of claim 24, wherein the chimeric B cell receptor is derived fromat least one of membrane IgG, CD19, BCMA, CD38, the common gamma chainreceptor, the IL-21 receptor, Toll-like Receptor, or CD40.
 26. Theisolated modified B cell of claim 24, wherein the cytotoxic effectormolecules include at least one of perforin, granzyme b, Fas ligand, ortumor necrosis factor-related apoptosis-inducing ligand (TRAIL).
 27. Theisolated modified B cell of claim 24, wherein the cytotoxic effectormolecules are expressed in response to a specific target cell or targetantigen.
 28. An isolated modified B cell capable of expressing: at leastone exogenously incorporated membrane immunoglobulin or recombinant Bcell receptor that is capable of binding to a first antigen; at leastone exogenously incorporated reassigned biological agent; and at leastone exogenously incorporated nucleic acid encoding secretedimmunoglobulin capable of binding to a second antigen.
 29. The isolatedmodified B cell of claim 28, wherein the at least one exogenouslyincorporated membrane immunoglobulin includes at least one exogenouslyincorporated membrane immunoglobulin polypeptide.
 30. The isolatedmodified B cell of claim 28, wherein the at least one exogenouslyincorporated membrane immunoglobulin is encoded by at least oneexogenously incorporated nucleic acid.
 31. The isolated modified B cellof claim 28, wherein the exogenously incorporated nucleic acid encodingthe at least one membrane immunoglobulin is integrated in one or morechromosomal loci in the isolated modified B cell.
 32. The isolatedmodified B of claim 31, wherein the chromosomal loci include at leastone of the Ig H chain or Ig L chain chromosomal loci.
 33. The isolatedmodified B cell of claim 32, wherein the Ig H chain or Ig L chainchromosomal loci are non-expressed Ig alleles.
 34. The isolated modifiedB cell of claim 32, wherein the Ig H chain or Ig L chain chromosomalloci are expressed Ig alleles.
 35. The isolated modified B cell of claim34, wherein at least one of the Ig H chain or Ig L chain chromosomalloci is under expression control of endogenous promoter or enhancerelements.
 36. The isolated modified B cell of claim 34, wherein the Ig Lchain locus includes at least one of the kappa or the lambda light(L)-chain locus.
 37. The isolated modified B cell of claim 36, whereinthe integration at the kappa or lambda light (L)-chain locus disruptsendogenous B cell kappa or lambda light (L)-chain expression. 38.(canceled)
 39. (canceled)
 40. (canceled)
 41. (canceled)
 42. (canceled)43. The isolated modified B cell of claim 32, wherein the at least oneexogenously incorporated membrane immunoglobulin nucleic acid includesan exogenous bicistronic expression construct.
 44. (canceled) 45.(canceled)
 46. (canceled)
 47. The isolated modified B cell of claim 28,wherein the at least one exogenously incorporated membraneimmunoglobulin includes a nucleic acid encoding one single chain Fvimmunoglobulin.
 48. The isolated modified B cell of claim 28, whereinthe isolated modified B cell includes at least one of naïve Blymphocyte, immature B lymphocyte, transitional B lymphocyte, mature Blymphocyte, B1 B lymphocyte, marginal zone B lymphocyte, follicular Blymphocyte, memory B lymphocyte, plasmablast, or plasma cell. 49.(canceled)
 50. (canceled)
 51. (canceled)
 52. The isolated modified Bcell of claim 28, wherein the membrane immunoglobulin includes at leastone of a membrane anchor, a cytoplasmic domain, a hinge region, or anextracellular ligand-binding domain.
 53. The isolated modified B cell ofclaim 28, wherein the isolated modified B cell bears at least onechimeric B cell receptor capable of transducing signals for inducingexpression of cytotoxic effector molecules.
 54. The isolated modified Bcell of claim 53, wherein the chimeric B cell receptor is derived fromat least one of membrane IgG, CD19, BCMA, CD38, the common gamma chainreceptor, the IL-21 receptor, Toll-like Receptor, or CD40.
 55. Theisolated modified B cell of claim 54, wherein the cytotoxic effectormolecules include at least one of perforin, granzyme B, Fas ligand, ortumor necrosis factor-related apoptosis-inducing ligand (TRAIL).
 56. Theisolated modified B cell of claim 54, wherein the cytotoxic effectormolecules are expressed in response to a specific target cell or targetantigen.
 57. The isolated modified B cell of claim 28, wherein the atleast one exogenously incorporated secreted immunoglobulin includes atleast one exogenously incorporated secreted immunoglobulin polypeptide.58. The isolated modified B cell of claim 57, wherein the at least oneexogenously incorporated secreted immunoglobulin is encoded by at leastone exogenously incorporated nucleic acid.
 59. The isolated modified Bcell of claim 58, wherein the exogenously incorporated nucleic acidencoding the at least one secreted immunoglobulin is integrated in oneor more chromosomal loci in the isolated modified B cell.
 60. Theisolated modified B cell of claim 59, wherein the chromosomal lociinclude at least one of the Ig H chain or Ig L chain chromosomal loci.61. The isolated modified B cell of claim 60, wherein the Ig H chain orIg L chain chromosomal loci are the non-expressed Ig alleles.
 62. Theisolated modified B cell of claim 59, wherein the Ig H chain or Ig Lchain chromosomal loci are the expressed Ig alleles.
 63. The isolatedmodified B cell of claim 62, wherein at least one of the Ig H chain orIg L chain chromosomal loci is under expression control of endogenouspromoter or enhancer elements.
 64. (canceled)
 65. (canceled) 66.(canceled)
 67. (canceled)
 68. (canceled)
 69. (canceled)
 70. (canceled)71. The isolated modified B cell of claim 59, wherein the at least oneexogenously incorporated secreted immunoglobulin nucleic acid includesan exogenous bicistronic expression construct.
 72. (canceled) 73.(canceled)
 74. (canceled)
 75. The isolated modified B cell of claim 59,wherein the at least one exogenously incorporated secretedimmunoglobulin includes a nucleic acid encoding one single chain Fvimmunoglobulin.
 76. An isolated modified B cell capable of expressing atleast one exogenously incorporated membrane immunoglobulin or arecombinant B cell receptor that is reactive to a first antigen; and atleast one endogenous secreted immunoglobulin reactive to a secondantigen; at least one exogenously incorporated reassigned biologicalagent; and at least one exogenously incorporated membrane receptorconfigured to directly or indirectly induce expression of at least onecytotoxic effector molecule.
 77. The isolated modified B cell of claim76, wherein the membrane receptor configured to directly or indirectlyinduce at least one cytotoxic effector molecule includes at least onechimeric receptor exhibiting at least a portion of at least one cytokinereceptor. 78.-87. (canceled)